Mission of GARA Annual GARA Scientific... · Effect of deleting African Swine Fever Virus...

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Mission of GARA

To establish and sustain global research partnerships that will generate scientific knowledge and tools to contribute to the successful prevention, control and where feasible eradication of African Swine Fever (ASF).

Vision of GARA

A coordinated global research all iance enabling the progressive control and eradication of ASF.

Strategic Goals of GARA

Goal 1. Identify research opportunities and facil itate collaborations within the Alliance

Goal 2. Conduct strategic and multi -disciplinary research to better understand ASF Goal 3. Determine social and economic drivers and impact of ASF Goal 4. Develop novel and improved tools to support the prevention and control of ASF Goal 5. Determine the impact of ASF prevention and control tools Goal 6. Serve as a communication and technology sharing gateway for the global ASF research community and stakeholders

Purpose of the Workshop

The main objective of the scientific workshop will be to review accomplishments made towards the gaps identified during the previous workshops on Plum Island and Pretoria. The topics will focus on virology and pathogenesis, epidemiology (Africa /

Europe), vaccines and immune responses, molecular epidemiology and diagnosis, legislation and reporting in different countries , use of genome editing to understand virus and host interaction.

Participants

Target audience is approximately 100 participants from previous GARA workshops on Plum Island and South Africa as well as

GARA partners and collaborators and other potential research partners. Invitees include scientific experts from European Union, Eastern Europe, Africa, Russia , Ukraine, Armenia and the Republic of Georgia, Australia, China, USA, Canada and representatives from international organizations, such as FAO, OIE, ILRI,…

Outputs of the workshop

The GARA will make use of the opportunities to identify research needs and establish collaborations and partnerships, update the gap analysis and identify potential funding sources to address those gaps. The Organizing Committee of the 3rd Annual GARA Scientific Workshop, together with the Scientific Committee, look forward to welcoming you in Ploufragan in September 2016 and assure that your participation will be fruitful and productive!

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Organizing Committee

Anses, Ploufragan-Plouzané Laboratory, France

Marie-Frédérique LE POTIER

Olivier BOURRY

Nicolas ROSE

Evelyne HUTET

Virginie LOISELIER

Anses, Department of Information, Communication and Dialogue with Society, France

Fabrice COUTUREAU

Sabine PUISEUX

Céline LETERQ

Executive committee

Cyril GAY, USDA Agricultural Research Service, USA

Melanie PETERSON, USDA Agricultural Research Service, USA

Bob ROWLAND, College of Veterinary Medicine, Kansas State University, USA

Covadonga ALONSO, INIA - Instituto Nacional de Investigación y Tecnología Agraria y Alimentaria, Spain

Scientific Commitee

Linda DIXON, The Pirbright Institute, United Kingdom

Marie-Frédérique LE POTIER, Anses, Ploufragan-Plouzané Laboratory, France

Livio HEATH, Onderstepoort Veterinary Institute, South Africa

Willie LOEFFEN, Central Veterinary Institute, Netherlands

Denis KOLBASOV, National institute for vet virology & microbiology, Russia

Eric ETTER, University Of Pretoria/CIRAD, South Africa

Charles MASEMBE, Makerere University, Uganda

Ana DE LA TORRE, INIA-CISA-Centro de Investigacion en Sanidad Animal Spain

Douglas GLADUE, USDA Agricultural Research Service, USA

Dolores GAVIER-WIDEN, SVA - National Veterinary Institute, Sweden

Armanda BASTOS, University of Pretoria, South-Africa

Carmina GALLARDO, INIA-CISA-Centro de Investigacion en Sanidad Animal, Spain

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AGENDA AND SCIENTIFIC PROGRAMME

Tuesday, 6th September 2016

8:15 Shuttle departure (Saint-Brieuc Zoopole Ploufragan)

8:15-8:45 Registration

8:45-9:00 Welcome Address Gilles SALVAT & Marie-Frédérique LE POTIER, France

9:00-9:45 General introduction: GARA presentation, main objectives and future Cyril GAY, USA

Scientific Session 1: “Virology & Pathogenesis” Chairs: Covadonga ALONSO, Spain – Douglas GLADUE, USA

9:45-12:45 Selected abstracts “Virology & Pathogenesis”

9:45 Natasha GAUDREAULT, USA Pigs lacking CD163 expression show no resistance to infection with African Swine Fever Virus.

10:00 Crystal JAING, USA

Whole Transcriptome Analysis of Pigs Infected with African Swine Fever Virus.

10:15 Douglas GLADUE, USA

The Ep152R ORF of African Swine Fever Virus strain Georgia encodes for an essential gene that

interacts with host protein BAG6.

10:30-11:15 Coffee Break & Poster Session

11:15 Covadonga ALONSO, Spain A new significance for African Swine fever cell targets at early infection.

11:30 Willie LOEFFEN, Netherlands

Effect of inoculation dose and age of the pigs on clinical, virological and serological parameters of

an African Swine Fever infection.

11:45 Laura ZANI, Germany

First evidence of an attenuated phenotype of genotype II African swine fever virus in Estonia.

12:00 Catharina KEßLER, Germany

Purification and Proteome analysis of African Swine Fever Virus Particles .

12:15 Günther KEIL, Germany

Adaptation of African Swine Fever Virus field strains to productively replicate in WSL cells is not

necessarily associated with larger deletions within the viral genomes.

12:30 -14:00 Lunch

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Scientific Session 2: “Vaccines and immune response” Chairs: Linda DIXON, United Kingdom - Marie-Frédérique LE POTIER, France

14:00-15:30 Selected abstracts “Vaccines and immune response”

14:00 Giulia FRANZONI, Italy

Response of monocyte-derived dendritic cells to virulent and attenuated ASFV strains .

14:15 Michael PARKHOUSE, Portugal Identification and util ity of interferon evasion by African Swine Fever Virus.

14:30 Linda DIXON, United Kingdom

Effect of deleting African Swine Fever Virus interferon inhibitory genes on virus pathogenesis and induction of protective responses in pigs.

14:45 Manuel BORCA, USA

Use of a double viral gene deletion virus as a rational approach to develop ASFV live attenuated

vaccines protecting against Georgia 2007 isolate.

15:00 Yolanda REVILLA-NOVELLA, Spain

Heterologous prime-boost vaccine strategy for ASF.

15:15 Fernando RODRIGUEZ, Spain

Could live-attenuated African Swine Fever Viruses become a vaccine reality?


16:00-17:00 Breakout Groups

17:00-18:00 Conclusion of the day – State of the Art

18:10 Shuttle (Zoopole Ploufragan Saint-Brieuc)

Wednesday, 7th September 2016


8:30-9:00 Registration

Scientific Session ASF-STOP: “Epidemiology” Chairs: Willie LOEFFEN, Netherlands – Dolores GAVIER-WIDEN, Sweden

9:00-9:20 Introduction ASF-STOP: objectives, working groups Dolores GAVIER-WIDEN, Sweden

9:20-9:50 Invited speaker presentation: Armanda BASTOS, South Africa

The African Swine Fever sylvatic cycle: using molecules and maths to unravel the role of the Ornithodoros

tick vector.

9:50-10:20 Keynote lecture: Daniel BELTRAN-ALCRUDO, FAO

The 1996-2002 African Swine Fever (ASF) pandemic in West Africa: Lessons learnt.

10:20- 10:40 Keynote lecture: Klaus DEPNER, Germany

Wild boar as ASF driver.

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11:10- 11:30 Keynote lecture: Krzysztof ŚMIETANKA, Poland

African Swine Fever in Poland - current situation and control measures

Scientific Session 3: “Epidemiology focusing in Europe” Chairs: Willie LOEFFEN, Netherlands – Dolores GAVIER-WIDEN, Sweden

11:30-12:45 Selected abstracts “Epidemiology focusing in Europe”

11:30 Imbi NURMOJA, Estonia

African Swine Fever Virus spread in Estonia: the preliminary results of epidemiological analysis .

11:42 Jan Hendrik FORTH, Germany Evaluation of insect larvae as possible mechanical vectors for transmission of ASFV in wild boar

populations.

11:54 Willie LOEFFEN, Netherlands

Quantification of transmission of African Swine Fever Virus by carriers and through indirect environmental contact.

12:06 Hans-Hermann THULKE, Germany

Active management of ASF spread in wild boar: strategic options and plausible solutions .

12:18 Martin LANGE, Germany

Towards quantitative understanding of the role of carcasses in the transmission of ASF in wild

boar or feral swine.

12:30 Serhii FILATOV, Ukraine

Research on African Swine Fever Threat Reduction Through Surveillance in the Ukraine: An

Update.

12:42 -14:00 Lunch

12:42 -14:00 Working lunch for ASF-STOP Core Group members (Core group meeting)

Scientific Session 4: “Epidemiology focusing in Africa” Chairs: Eric ETTER, South Africa – Charles MASEMBE, Uganda

14:00-14:30 Keynote Lecture: Charles MASEMBE, Uganda Wild life and domestic livestock interactions in endemic situation.

14:30-15:30 Selected abstracts “Epidemiology focusing in Africa”

14:30 Mary-Louise PENRITH, South Africa

Epidemiology of African Swine Fever in Africa today: Sylvatic cycle versus socio-economic

imperatives – fi l l ing the gaps .

14:45 Erika CHENAIS, Sweden

Quantitative assessment of socio-economic impacts of African Swine Fever outbreaks in

northern Uganda.

15:00 Peter OGWENG, Uganda A molecular and ecological investigation of the role of the bushpig in the epidemiology of

African Swine Fever at the wildlife-livestock interface in Uganda.


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15:30-17:00 Parallel Meeting ASF-STOP Core Group members (Core group meeting-continuation)

16:00 Laurence VIAL, France

Experimental versus natural determinants influencing the vector competence of Ornithodoros soft ticks for African Swine Fever Virus .

16:15 Ariane PAYNE, Uganda

The potential role of the bushpig (Potamochoerus larvatus) in the spread and maintenance of

African Swine Fever.

16:30 Edward OKOTH-ABWORO, Kenya

Integrated study of African Swine Fever in East Africa identify the disease drivers and provides

insights for targeted surveillance and control .

17:00-17:40 Breakout Groups / Breakout Group ASF-STOP

17:40-18:00 Conclusion of the day-State of the Art

18:10

19:00

Shuttle (Zoopole Ploufragan Gala dinner)

GALA DINNER (best poster prize)

Thursday, 8th September 2016


9:00-9:30 GARA future perspectives : Cyril GAY, USAElection new committee

Scientific Session 5: “Molecular Epidemiology & Diagnosis” Chairs: Carmina GALLARDO, Spain – Armanda BASTOS, South Africa

9:30-10:30 Selected abstracts “Molecular Epidemiology & Diagnosis”

9:30 Silvia DEI GIUDICI, Italy

Molecular characterization of Sardinian African Swine Fever Viruses based on analysis of p30,

CD2V and I73R/I329L variable region.

9:45 Juanita VAN HEERDEN, South Africa African Swine Fever (ASF) genotype II in Zimbabwe during 2015.

10:00 Pam LUKA, Nigeria

Detection of antibodies to Ornithodoros saliva antigen in domestic pigs from Cross River and Taraba.

10:15 Clara YONA, TanzaniaEpidemiological study and socio-economic impact associated with persistence of African swine fever in Southern highlands of Tanzania.


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11:15 Emmanuel AWOSANYA, Nigeria

A new African Swine Fever Virus strain of genotype I circulates among pig herds in southwest

Nigeria.

11:30 David WILLIAMS, Australia

Rapid detection and epidemiological surveillance of African Swine Fever using oral fluid.

11:45 Stella ATIM, Uganda

Evaluation of a portable real -time PCR platform (TCOR-8) for ASF during outbreaks in an endemically infected population in Uganda.

12:00-12:45 Conclusion of the day -State of the Art

12:45 -14:00 Lunch

Scientific Session 6: “Legislation and reporting in different countries” Chairs: Ana DE LA TORRE, Spain – Denis KOLBASOV, Russia

14:00-15:30 Introduction of discussion: FAO/EFSA/EC/OIERound table discussion with experts from different regions all over the world


16:00-17:00 Breakout Groups



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TABLE OF CONTENTS

Scientific Session 1: “Virology & Pathogenesis”

Natasha GAUDREAULT, USA ..................................................................................................................... 16 Pigs lacking CD163 expression show no resistance to infection with African Swine Fever Virus

Crystal JAING, USA .................................................................................................................................... 16 Whole Transcriptome Analysis of Pigs Infected with African Swine Fever Virus

Douglas GLADUE, USA .............................................................................................................................. 17 The Ep152R ORF of African Swine Fever Virus strain Georgia encodes for an essential gene that interacts with host protein BAG6

Covadonga ALONSO, Spain ....................................................................................................................... 17 A new significance for African Swine Fever cell targets at early infection

Willie LOEFFEN, Netherlands..................................................................................................................... 18

Effect of inoculation dose and age of the pigs on clinical, virological and serological parameters of an African Swine Fever infection

Laura ZANI, Germany................................................................................................................................ 18 First evidence of an attenuated phenotype of genotype II African Swine Fever Virus in Estonia

Catharina KEßLER, Germany ..................................................................................................................... 19

Purification and Proteome analysis of African Swine Fever Virus Particles

Günther KEIL, Germany............................................................................................................................. 20

Adaptation of African Swine Fever Virus field strains to productively replicate in WSL cells is not necessarily associated with larger deletions within the viral genomes

Scientific Session 2: “Vaccines and immune response”

Giulia FRANZONI, Italy.............................................................................................................................. 22

Response of monocyte-derived dendritic cells to virulent and attenuated ASFV strains

Michael PARKHOUSE, Portugal ................................................................................................................. 22 Identification and util ity of interferon evasion by African Swine Fever Virus

Linda DIXON, United Kingdom................................................................................................................... 23

Effect of deleting African Swine Fever Virus interferon inhibitory genes on virus pathogenesis and induction

of protective responses in pigs

Manuel BORCA, USA................................................................................................................................. 23

Use of a double viral gene deletion virus as a rational approach to develop ASFV live attenuated vaccines protecting against Georgia 2007 isolate

Yolanda REVILLA-NOVELLA, Spain ............................................................................................................. 24

Heterologous prime-boost vaccine strategy for ASF

Fernando RODRIGUEZ, Spain .................................................................................................................... 24


Scientific Session ASF-STOP: “Epidemiology”

Armanda BASTOS, South Africa ................................................................................................................. 27

The African Swine Fever sylvatic cycle: using molecules and maths to unravel the role of the Ornithodoros tick vector

Daniel BELTRAN-ALCRUDO, FAO ............................................................................................................... 28

The 1996-2002 African Swine Fever (ASF) pandemic in West Africa: Lessons learnt

Klaus DEPNER, Germany ........................................................................................................................... 29

Wild boar as ASF driver

Krzysztof ŚMIETANKA, Poland................................................................................................................... 30

African Swine Fever in Poland - current situation and control measures

Scientific Session 3: “Epidemiology focusing in Europe”

Imbi NURMOJA, Estonia............................................................................................................................ 32 African Swine Fever Virus spread in Estonia: the preliminary results of epidemiological analysis

Jan Hendrik FORTH, Germany ................................................................................................................... 32 Evaluation of insect larvae as possible mechanical vectors for transmission of ASFV in wild boar populations

Willie LOEFFEN, Netherlands..................................................................................................................... 33

Quantification of transmission of African Swine Fever Virus by carriers and through indirect environmental contact

Hans-Hermann THULKE, Germany ............................................................................................................. 33

Active management of ASF spread in wild boar: strategic options and plausible solutions.

Martin LANGE, Germany........................................................................................................................... 34

Towards quantitative understanding of the role of carcasses in the transmission of ASF in wild boar or feral swine

Serhii FILATOV, Ukraine ........................................................................................................................... 34Research on African Swine Fever Threat Reduction Through Surveillance in the Ukraine: An Update

Scientific Session 4: “Epidemiology focusing in Africa”

Charles MASEMBE, Uganda ..................................................................................................................... 36 Wild life and domestic l ivestock interactions in endemic situation

Mary-Louise PENRTH, South Africa ............................................................................................................ 37

Epidemiology of African Swine Fever in Africa today: Sylvatic cycle versus socio-economic

imperatives – fi l l ing the gaps

Erika CHENAIS, Sweden............................................................................................................................. 37 Quantitative assessment of socio-economic impacts of African Swine Fever outbreaks in northern Uganda

Peter OGWENG, Uganda .......................................................................................................................... 38 A molecular and ecological investigation of the role of the bushpig in the epidemiology of African Swine Fever at the wildlife-livestock interface in Uganda

Laurence VIAL, France............................................................................................................................... 39

Experimental versus natural determinants influencing the vector competence of Ornithodoros soft ticks

for African Swine Fever Virus

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Ariane PAYNE, Uganda............................................................................................................................. 39 The potential role of the bushpig (Potamochoerus larvatus ) in the spread and maintenance of African Swine Fever

Edward OKOTH-ABWORO, Kenya.............................................................................................................. 40

Integrated study of African Swine Fever in East Africa identify the disease drivers and provides insights for targeted surveillance and control

Scientific Session 5: “Molecular Epidemiology & Diagnosis”

Silvia DEI GIUDICI, Italy ............................................................................................................................. 42 Molecular characterization of Sardinian African Swine Fever Viruses based on analysis of p30, CD2V

and I73R/I329L variable region

Juanita VAN HEERDEN, South Africa .......................................................................................................... 42 African Swine Fever (ASF) genotype II in Zimbabwe during 2015

Pam LUKA, Nigeria.................................................................................................................................... 43

Detection of antibodies to Ornithodoros saliva antigen in domestic pigs from Cross River and Taraba

Clara YONA, Tanzania .............................................................................................................................. 43 Epidemiological study and socio-economic impact associated with persistence of African swine fever in Southern highlands of Tanzania. Emmanuel AWOSANYA, Nigeria................................................................................................................ 44

A new African Swine Fever Virus strain of genotype I circulates among pig herds in southwest Nigeria

David WILLIAMS, Australia ....................................................................................................................... 44

Rapid detection and epidemiological surveillance of African Swine Fever using oral fluid

Stella ATIM, Uganda ................................................................................................................................ 45

Evaluation of a portable real -time PCR platform (TCOR-8) for ASF during outbreaks in an endemicall y infected population in Uganda

Poster Session

P01 Maria Vittoria MURGIA, USA ........................................................................................................ 48

Development of monoclonal antibodies against ASFV major structural proteins: p72, p54, and p30

P02 Jolene CARLSON, USA ................................................................................................................... 48

Dynamics of protection against virulent challenge in swine vaccinated with attenuated African Swine Fever

Viruses and their differences in pathogenesis and immune responses

P03 David CHAPMAN, United-Kingdom................................................................................................ 49

Comparative study of protection in pigs immunised by different routes and doses with the naturally

attenuated African Swine Fever Virus isolate OUR T88/3 and role of immunomodulatory cytokines

P04 Shawn BABIUK, Canada ................................................................................................................ 49

Evaluation of porcine adenovirus vectored vaccines for African Swine Fever

P05 Marie-Frédérique LE POTIER, France.............................................................................................. 50

Is Ornithodoros erraticus able to transmit the Georgia2007/1 African Swine Fever Virus isolate to domestic pigs?

P06 Thibaud PORPHYRE, United-Kingdom ............................................................................................ 50

Research facil itation of pig sector and government preparedness for exotic incursions of swine fevers:

the case of Scotland

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P07 Sandro ROLESU, Italia ................................................................................................................... 51

The role of socio-economic factors on African Swine Fever in Sardinia

P08 Ioseb MENTESHASHVILI, Georgia .................................................................................................. 51

African Swine Fever Risk Assessment and Preparedness in Georgia

P09 Tengiz CHALIGAVA, Georgia .......................................................................................................... 52

ASF Public Outreach Program in Georgia

P10 Roman DATSENKO, Ukraine .......................................................................................................... 52

Raising awareness of farmers and veterinarians for efficient respond to African Swine Fever in Ukraine

P11 Ana Isabel DE LA TORRE, Spain...................................................................................................... 53

Quality of available habitats for wild boar as a online cartographic tool for managing African Swine Fever

in Eurasia

P12 Laura MEIER, Germany ................................................................................................................. 53

Integrating ASF sero-prevalence data in wild boar with the decreased-virulence-hypothesis

P13 Franck KOENEN, Belgium............................................................................................................... 54

Evaluation of the efficiency of passive surveillance in the early detection of African Swine Fever in the wild boar

P14 Yiltawe Simwal WUNGAK, Nigeria ................................................................................................ 54

Serological analyses of African Swine Fever in Nigeria, 2006-2015

P15 Erika CHENAIS, Sweden ................................................................................................................. 55

Biosecurity breaches and within-farm virus contamination during an African Swine Fever outbreak

on a medium sized farm in Uganda

P16 Carin BOSHOFF, South Africa ........................................................................................................ 55

Phylogeography of the African Swine Fever ornithodoros vectors in Southern Africa

P17 Noelina NANTIMA, Uganda .......................................................................................................... 56

Socio-economic impact of African Swine Fever outbreaks in smallholder pig systems in four districts

along the Uganda-Kenya border

P18 Edward OKOTH-ABWORO, Kenya .................................................................................................. 56

Consultation on a research strategy for the implementation of a regional control program for

African Swine Fever in Africa

P19 Andrzej KOWALCZYK, Poland ........................................................................................................ 57

Polymerase cross-linking spiral reaction (PCLSR) for detection of African Swine Fever Virus (ASFV)

in pigs and wild boars

P20 Silvia DEI GIUDICI, Italy ................................................................................................................. 57

Genomic analysis of Sardinian 26544/OG10 isolate of African Swine Fever Virus

P21 Juanita VAN HEERDEN, South Africa .............................................................................................. 58

A universal protocol to generate consensus level genome sequence for African Swine Fever Viruses

P22 Gian Mario DE MIA, Italy .............................................................................................................. 58

Development of a DNA/LNA optical biosensor for the rapid diagnosis of African Swine Fever

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P23 Gian Mario DE MIA, Italy .............................................................................................................. 59

Complete genome sequencing of Sardinian African Swine Fever virus isolates

P24 Oleg NEVOLKO, Ukraine ................................................................................................................ 59

Molecular analysis of African Swine Fever Virus (ASFV) associated with disease outbreaks in Ukraine

during 2012-2015

P25 Johnson Francis MAYEGA, Uganda................................................................................................ 60

The challenges encountered in diagnosing African Swine Fever in Uganda: are positives truly positive?

P26 Vincent MICHAUD, France............................................................................................................. 60

Molecular characterization of Malagasy African Swine Fever Virus strains from 2008 to 2014

P27 Simona PILEVICIENE, Lithuania ...................................................................................................... 61

African Swine Fever situation in Lithuania

P28 Loïc COMTET, France..................................................................................................................... 61

Validation of new Elisa & PCR for the diagnosis of African Swine Fever

P29 Mathias AFAYOA, Uganda ............................................................................................................ 62

Application of chicken egg yolk anti African Swine Fever viral protein vp73 IgY in a card agglutination

test for sero-diagnosis of African swine fever

List of participants .................................................................................................. 63

Dear colleagues

On behalf of the French Agency ANSES and the GARA executive committee, it is my great pleasure to welcome you in PLOUFRAGAN for the 3rd GARA workshop. As you know this initiative was launched the 1st time in Plum-Island by Cyril Gay and the second

workshop took place in Pretoria. So we can say that GARA has already visited three continents.

Today, all continents are represented. We are 135 scientists coming from 30 countries strive for the

same goal: to control the African swine fever!

68 abstracts were submitted from which 39 oral presentations and 29 poster exhibitions were

accepted.

I would like to thank Anses that supported financially the organization of this workshop; DTRA-CBEP

(Defense Threat Reduction Agency – Cooperative Biological) & CDRF Global for their travel grants for

scientist coming from affected countries (Africa or East Europe); and ASF-STOP, an e- COST action

that I invited to join this workshop as we have many common objectives between GARA and ASF-

STOP. Her coordinator Dolores Gavier-Widen helped in its preparation and by giving also grant

travels for invited speakers.

And finally I would like to thank the Scientific and Local Organizing Committees for their commitment and activities to set up this event, who helped me a lot in the organization of this workshop with a special dedication to Virginie, Melanie and Cyril! Cyril Gay will explain longer the main objectives of GARA, but I would like to remember you that we

are all together here to identify the research gaps on the path to the control of the disease and

propose future orientations. This can be achieved during the workshop because of your highly active

participation to the breakout groups that will be organized every day by the chairs.

Every day, we‘ll have two different scientific sessions and time reserved for the poster session. One

poster will be rewarded during the gala dinner. On the last day, we’ll have a round table on ASF

reporting and legislation. Representatives from international bodies as OIE, FAO and EFSA accepted

to participate with representatives of affected countries. This workshop has been organized by Ana

de la Torre and Denis Kolbasov, and I am sure that will be of high interest for all of us.

Finally, I wish for everybody, a successful and productive congress, a good stay in our beautiful area and some opportunities to visit a highly attractive region. Enjoy the congress, enjoy Brittany, and enjoy our lifestyle!

Marie-Frédérique Le Potier Chair Scientific and Organizing Committees

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Chairs:

Covadonga ALONSO, Spain Douglas GLADUE, USA

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Pigs lacking CD163 expression show no resistance to infection with African Swine Fever

Virus

Natasha Gaudreault1, Kristin M. Whitworth

2, Luca Popescu

1, Maria Murgia

1, Jerome Nietfeld

1, Alan Mileham

3, Melissa

Samuel2, Kevin D. Wells

2, Randall S. Prather

2 and Raymond R.R. Rowland

1

1Diagnostic Medicine and Pathobiology, College of Veterinary Medicine, Kansas State University, Manhattan, KS 66506, U.S.A.; 2Division of

Animal Science, College of Food Agriculture and Natural Resources, University of Missouri, Columbia, MO 65211, U.S.A.; 3Genus, plc,

DeForest, Wisconsin, U.S.A.

Keywords: CD163, African swine fever virus, Georgia 2007/1

The macrophage surface protein, CD163, has been implicated as a receptor for the African swine fever virus (ASFV). Yet

support for that model is based on in vitro laboratory experiments. We recently evaluated the response of pigs, genetically

edited to knockout CD163, to infection with ASFV. Following infection with the virulent Georgia 2007/1 isolate, a cohort of

wild type and CD163 knockout pigs showed no differences in clinical signs, pathology or viremia. Infection of macrophages in

vitro exhibited similar results. This data clearly demonstrate that CD163 is not required for infection with the Georgia isolate,

and that future research should focus on other possible receptors and entry pathways. Understanding these mechanisms

and virus-host interactions will be important for designing better antiviral strategies against this disease.

Whole transcriptome analysis of pigs infected with African Swine Fever Virus

Crystal Jaing1, Jonathan Allen

2, James B. Thissen

1, Raymond R.R. Rowland

3, David Will iams

4

1Physical & Life Sciences Directorate - 2Computations Directorate, Lawrence Livermore National Laboratory, Livermore, California, USA - 3Department of Diagnostic Medicine and Pathobiology, Kansas State University, Manhattan, Kansas, USA -

4Australian Animal Health

Laboratory, Geelong, Australia

Keywords: RNAseq, immune response, vaccine, diagnostic, pathogenesis

There is a significant need to better understand the mechanisms of African swine fever virus (ASFV) infection in pigs to aid

the development of countermeasures and diagnostics. We pursued a genome-wide approach to characterize the pig immune

responses after ASFV infection. Pigs were infected via the oronasal route with either the low pathogenic OURT88/3 strain, or

the highly pathogenic GRG2007 strain. Blood samples were collected at day 0 and at euthanasia day (ED) for infections with

both strains, and on 3, 7, 14 and 28 days post-infection (dpi) with OURT88/3 strain. No clinical signs or lesions at post-

mortem were observed in pigs infected with OURT88/3, whereas si gns and lesions in pigs infected with the GRG2007 were

characteristic of highly pathogenic ASFV. Four of six pigs infected with OURT88/3 seroconverted, while none of the GRG2007 -

infected pigs were antibody positive, consistent with the acute disease course. High levels of virus genome and antigen were

detected in multiple organs of the GRG2007-infected pigs, with highest levels in the lymphoid tissues. RNAseq analysis of

whole blood samples identified 395 genes most differently expressed at ED (7 -10 dpi) in the GRG2007 group, and 181 genes

at 7 dpi in the OURT88/3 group. We also identified a set of common genes that are most significantly changed with both the

OURT88/3 and the GRG2007 group including macrophage markers, natural kil ler cell markers, chemokines and other

important immune response markers. Viral pathogenesis, RNAseq, gene expression and pathway analyses from this study will

be presented.

RETURN

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The Ep152R ORF of African Swine Fever Virus strain Georgia encodes for an essential

gene that interacts with host protein BAG6

Manuel V. Borca, Vivian O’Donnell, Lauren G. Holinka, Devendra K. Rai, Brenton Sanford, Marialexia Alfano, Jolene Carlson, Paul A. Azzinaro and Douglas P. Gladue

Agricultural Research Service and Department of Homeland Security, Plum Island Animal Disease Center, Greenport, NY 11944, USA

Department of Pathobiology and Veterinary Science, University of Connecticut, Storrs, CT 06269, USA Oak Ridge Institute for Science and

Education (ORISE), Oak Ridge, TN 37831 Biosecurity Research Institute and Department of Diagnostic Medicine and Pathobiology, College of

Veterinary Medicine, Kansas State University, Manhattan

Keywords: ASFV, Viral-Host protein interaction, EP152R

African swine fever virus (ASFV) is the etiological agent of a contagious and often lethal disease of domestic pigs that has significant

economic consequences for the swine industry. The viral genome encodes for more than 150 genes, and only a select few have

been studied in some detail. Here we report the characterization of open reading frame Ep152R predicted to have a complement

control module/SCR domain which similar to Vaccinia virus proteins involved in blocking the immune response during viral

infection. A recombinant ASFV harboring a HA tagged version of the EP152R protein was developed (ASFV-G-EP152R-HA) and used

to demonstrate that EP152R is an early virus protein. Attempts to construct recombinant viruses having a deleted EP152R gene,

were consistently unsuccessful indicating that EP152R is an essential gene. Interestingly, analysis of host-protein interactions

between EP152R with using a yeast two-hybrid screen, identified BAG6, a protein previously identified as being required for ASFV

replication. Furthermore, fluorescent microscopy analysis confirms that EP152R-BAG6 interaction actually occurs in cells infected

with ASFV.

A new significance for African Swine Fever cell targets at early infection

Cuesta-Geijo, Miguel Angel, Raquel Muñoz-Moreno, Lucía Barrado-Gil, Inmaculada Galindo, Sergio Viedma, Covadonga

Alonso

Dpt. Biotecnología (INIA)

Keywords: ASFV entry, ASFV endosomal entry, Viral fusion, Cholesterol efflux, Interferon-induced proteins

We searched for African swine fever virus cell targets at early infection stages in order to find potential restriction factors at the

endosomal entry pathway. These celular restriction factors might be used to boost innate immunity and to develop intervention

strategies against the virus. ASFV traffics through the endocytic pathway where uncoating takes place at the acidi c pH of the

multivesicular endosomes. After uncoating, ASFV internal membrane fuses with the endosomal membrane to allow cytoplasmic exit

of virions to start replication. Cholesterol plays an essential role in the establishment of ASFV infection and is required for this viral

fusion step. In fact, viral fusion of ASFV, requires the integrity of cholesterol endosomal efflux, in contrast to other DNA viruses,

such as vaccinia virus or adenovirus 5 which are independent of this flux. In ASFV, the accumulation of cholesterol impairs viral exit

to the cytoplasm, resulting in retention of virions inside endosomes. This mechanism might be targeted by the innate immunity

restriction proteins, called InterFeron-Induced TransMembrane proteins (IFITMs). IFITMs act at the endosomal membrane impairing

viral fusion. This highlights the role of cholesterol at the start of viral replication. ASFV remodels intracelular cholester ol by

increasing its cellular uptake and redistributes free cholesterol to viral replication sites. Our analysis reveals that ASFV manipulates

cholesterol dynamics to surpass innate immunity in order to ensure an appropriate l ipid flux to establish productive infectio n.

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Effect of inoculation dose and age of the pigs on clinical, virological and serological

parameters of an African Swine Fever infection

E. Weesendorp, P.L. Eblé, H.W.M. Moonen-Leusen, S. Quak, W.L.A. Loeffen

Central Veterinary Institute of Wageningen UR (CVI), Lelystad, The Netherlands

Keywords: ASF, virus, age, dose, infection

Experimental infections with ASFV are mostly carried out in young piglets only. Under field conditions all different age categories

are at risk and individual pigs may be exposed to very different doses of virus. The aim of this study was to investigate th e possible

effect of ASFV inoculation dose and age of the pigs on clinical, virological and serological parameters. Four groups of five pigs each

were used in this study. Two groups (A and B) were 12 weeks old. The other two groups (C and D) were 18 weeks old. All pigs were

inoculated with ASFV (Netherlands ’86, a moderately virulent strain) intranasally. Groups A and C were inoculated with a rela tively

low dose (2ml of 10^3.5TCID50/ml) while groups B and D were inoculated with a relatively high dose (2ml o f 10^6 TCID50/ml). Pigs

were subsequently observed for 55 days (body temperatures, clinical signs, EDTA and serum blood samples, OPF and air samples ).

Age and infection dose had no or only a very l imited effect on the investigated parameters. However, mor e of the older piglets

survived the ASF infection and became carriers (five old pigs, vs. one young pig) with a lower maximum clinical score in the

survivors. Increased survival times for older pigs will l ikely result in shedding more virus into the enviro nment. Older pigs may

therefore play a more important role in transmission of the virus. The carriers still shed virus and showed a viraemia after 48-55 dpi,

without showing clinical signs of ASF. The extent to which these carriers were able to transmit th e virus was investigated in a

separate study.

First evidence of an attenuated phenotype of genotype II African Swine Fever Virus in

Estonia

Zani Laura1; Nurmoja Imbi

2; Breidenstein Christiane

1; Leidenberger Simone

1; Beer Martin

1; Blome Sandra

1

1Friedrich-Loeffler-Institut, Greifswald – Insel Riems, Germany - 2Estonian Veterinary and Food Laboratory, Tartu, Estonia

Keywords: African swine fever virus, attenuation, pathogenesis, characterization

African swine fever (ASF) is a severe, multi-systemic disease of pigs that is caused by the eponymous double-stranded DNA virus

(ASFV) of the genus Asfivirus within the Asfarviridae family. In 2014, ASF was re-introduced into the European Union, affecting

domestic pigs and wild boar in the Baltic States and Poland. Up to now, new cases have been reported especially from wild boar

every week. However, the geographic region remains rather stable. This epidemiological behaviour was rather unexpected based on

the experimental findings that the virus strains involved showed exceptionally high virulence. It was anticipated that the virus would

either spread rapidly or die out due to self-l imitation. Factors leading to the observed long-term outbreak are far from being

understood. One explanation could be virus attenuation. In fact, wild boar hunted in North-Eastern Estonia were tested ASFV

antibody positive without showing obvious clinical or pathological signs. To explore the reason for this phenomenon, a re-isolated

Estonian virus was biologically tested in different experiments. In a first experiment, ten sub-adult wild boar were inoculated. Here,

the virus showed high virulence, and nine out of ten animals succumbed to infection showing typical lesions. Subsequently, a virus

re-isolated from the recovered animal was utilized in two trials with different pig breeds. Trial A comprised 12 minipigs, trial B five

domestic fattening pigs. In both trials, oronasal inoculation was carried out using a blood suspension containing at least 10 4.5

hemadsorbing units. Upon infection, clinical signs were recorded daily. Furthermore, blood and swab samples were taken at regular

intervals. Necropsy was carried out on all animals and organ samples were collected. Blood, swab and organ samples were

subjected to PCR analyses and virus isolation, serum samples were analyzed using commercial ELISA kits and indirect

immunoperoxidase test. In trial A, all animals developed fever and unspecific clinical signs within the first week post inoc ulation.

However, nine out of twelve minipigs survi ved the acute phase and were slaughtered in good health status at 36 days post

inoculation (DPI). One animal was found dead after blood sampling and two others were euthanized due to severe respiratory

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distress. These deaths were not clearly linked to the disease course. Necropsy revealed that two of the inoculated minipig sows

were pregnant. Organ pools of the fetuses were tested by PCR and were found negative. Antibodies were detected in all

convalescent animals by the end of the trial. Animals of trial B displayed a similar disease course with all animals surviving ti l l the

end of the trial at 36 DPI. Apart from a severe fibrinous pericarditis in one of the animals, necropsy did not reveal any sig ns

indicative for ASF or any other disease. While clinical signs were completely absent from 19 DPI, high ASFV genome loads were stil l

detectable by PCR til l the end of the trial. Summarizing, an apparently attenuated virus strain was re-isolated from the initial trial.

Animals of different pig breeds, i .e. minipigs and domestic fattening hybrids, showed similar disease courses that were, in terms of

virus detection, still comparable to the highly virulent ancestors. However, clinical signs and mortality were drastically reduced.

Under field conditions, these unspecific clinical signs could easily go unnoticed and thus complicate disease control tremendously.

For the wild boar situation in Estonia, circulation of attenuated strains is l ikely and should be further investigated.

Purification and Proteome analysis of African Swine Fever Virus Particles

Keßler Catharina¹; Keil Günther¹; Blome Sandra²; Karger Axel¹

¹Friedrich Loeffler Institut Federal Research Institute for Animal Health, Institute of molecular Virology and Cell Biology ²Institute of Virus

Diagnostics, Greifswald-Island of Riems-Germany;

Keywords: ASFV particles, proteome analysis, mass spectrometry

African swine fever (ASF) is a viral disease that affects members of the Suidae family such as bush pigs, warthogs, domestic

pigs, and wild boars and is transmitted by direct contact with infected animals or by soft ticks of the Ornithodoros genus.

Warthogs and bush pigs are generally asymptomatic and are wildlife reservoirs for the virus in Afrika. While ASF had long

been endemic in sub-Saharan Africa and in Sardinia, the recent spread into Trans -Caucasian countries has raised concern as

ASF severely threatens the European pig industry. In spite of this fact, l iterature on virus -host interactions that could be used

as targets for antiviral strategies is scarce. As the morphogenesis of ASFV is very complex and leads to mature enveloped

intracellular virus particles and extracellular virions that carry an additional envelope, the preparation of pure extracellu lar

virions is challenging. To date, highly purified ASF virion preparations have not been analyzed systematically using modern

mass spectrometric approaches. The intention of this study is to define the proteome of the ASFV particle and to identify

virus-host interactions that potentially pl ay a role in virus morphogenesis. This work describes the purification of ASFV

particles and the characterization of the proteome of mature extracellular ASF virions using shotgun nLC -MALDI-TOF/TOF

mass spectrometry. A convenient and efficient workflow for the purification of ASFV particles has been established. ASFV

OUR T88/3 was propagated on WSL-HP cells which originate from a natural host (wild boar) using high-density cell culture

flasks with 10 layers (HYPERFlask, Corning) to achieve high virus yields. Particles were purified by a combination of

differential sedimentation and gradient density ultracentrifugation steps using sucrose and Iodixanol (OptiPrep) as gradient

media. Separation was based on particle density as well as on particle dynamics resu lting in preparations with high specific

infectivity. Use of Iodixanol facil itated the purification of virus, probably due to its low viscosity. Total virus recovery was

between 35-70 % after the gradient step. The final virion preparation was characterized by electron microscopy, western blot

analysis using virus-specific proteins, and finally by shotgun nLC-MALDI-TOF/TOF mass spectrometry. We present a

comprehensive catalog of viral and host-derived structural ASFV proteins.

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Adaptation of African Swine Fever Virus field strains to productively replicate in WSL

cells is not necessarily associated with larger deletions within the viral genomes.

Keil Günther M.1; Goller Katja

1; Pollin Reiko

1; Bishop Richard

2; Höper Dirk

1; Blome Sandra

1; Portugal Raquel

1

1Friedrich-Loeffler-Institut, Greifswald Insel Riems, Germany - 2International Livestock Research Institute (ILRI), Nairobi, Kenya

Keywords: ASFV field isolates, adaptation to replicate in WSL cells, NGS sequencing

The genomes of ASFV isolates are double stranded DNA and vary in size from 170 - 193 kbp. The genomes sizes vary, strain

dependent, between 170 and 190 kilo base pairs. The encompass 151 - 167 open reading frames in the range from 151 to

167. Although significance and functions for many genes have been studied so far, much more stil l need elucidation. For

doing that, cell culture adaptation appears to be a prerequisite since the natural host cells, monocytes and macrophages, are

difficult to work with as primary cultures. In the past, this process was frequently associated with considerable genome

modifications including large deletions. We adapted field isolates Armenia 2007, Benin 97, Kenya1033, and Sardinia 2015) to

productively grow in WSL cells. Full protein coding genome sequences were so far determined for Benin 97, Kenya1033, and

Sardinia by NGS. For the present, sequence comparisons revealed that the genomes of the respective WSL-adapted virus do

not contain larger deletions by comparison to respective or related field strains or to other virus adaptations to Vero cells.

Detailed analyses will be presented.

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Chairs:

Linda DIXON, United Kingdom Marie-Frédérique LE POTIER, France

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Response of monocyte-derived dendritic cells to virulent and attenuated ASFV strains

Giulia Franzoni 1,2

, Simon P. Graham3, Antonio G. Anfossi

1, Giovannantonio Pilo

2, Piero Bonelli

2, Marco Pittau

1, Paola

Nicolussi2, Si lvia Dei Giudici

2, Annalisa Oggiano

2.

1Department of Veterinary Medicine, University of Sassari, Sassari, 07100, Italy. - 2Istituto Zooprofilattico Sperimentale della Sardegna,

Sassari, 07100, Italy. - 3 School of Veterinary Medicine, University of Surrey, Guildford, GU2 7XH, United Kingdom.

Keywords: ASFV, dendritic cells, flow cytometry, cytokines

African swine fever virus (ASFV) primarily infects cells of the myeloid l ineage and despite the important role of dendritic c ells

(DCs) in initiating adaptive immune response to pathogens, few studies have analysed their interactions with this virus. Our

study therefore aimed to conduct a detailed characterization of the interactions between DCs and ASFV strains of differing

virulence. Isolated porcine peripheral blood monocytes were cultured in the presence of GM-CSF and IL-4 to induce

differentiation into immature DCs (MoDC). MoDC were left untreated or matured with TNF-α and/or IFN-α. Cells were

infected with an attenuated strain (BA71V) or a virulent Sardinian isolate (22653/14) of ASFV or were mock-infected. ASFV

infection and its consequence on MoDC phenotype and function are being analysed using flow cytometry, ELISA and confocal

microscopy. ASFV 22653/14 presented a greater ability to infect both immature and mature MoDC compared to the avirul ent

strain. Lower levels of late protein p72 but not early protein p30 were observed in BA71V-infected MoDCs pre-treated with

IFN-α, but not TNF-α, while 22653/14 retained the ability to infect IFN-α matured MoDC. Cells infected with either strain

displayed a lower expression of CD16 compared to uninfected bystander cells and BA71V-infected cells presented lower

percentages of MHC I compared to the mock-infected control. Ongoing experiments investigating the effects of ASFV on

other surface markers express ion and cytokine responses by MoDCs will be presented. It is hoped that data generated by this

study will aid our understanding of the immunomodulation of host cell responses by ASFV.

Identification and utility of interferon evasion by ASFV

Robert Michael Parkhouse

Instituto Gulbenkian de Ciência, Oeiras 2781-901, Portugal

Keywords: Interferon, non-homologous virus evasion genes

The interferon (IFN) system is an early innate anti -virus host defense mechanism that takes place shortly after entry of the

pathogen and long before the onset of adaptive immunity. Thus, African swine fever virus (ASFV), as an acute and persistent

virus in pigs, is predicted to have evolved multiple genes for the manipulation and evasion of interferon. Although, ASFV is

known to interfere with signaling pathways controlling the transcription of cytokines, surprisingly no individual virus gene

manipulating the induction or impact of IFN has been described. Since an initial bioinformatics search of the ASFV genome

failed to identify potential antagonists of the IFN response, our strategy was to functionally screen “unassigned” ASFV genes

without existing homologies, particularly from MGFs 360 and 530, in luciferase reporter assays for their inhibition of the

induction and impact of IFN. Specifically, we used reporter plasmids containing the luciferase gene under the control of: 1)

The IFN-β promoter, to screen for inhibition of induction of type I IFN stimulated by the addition of Poly(I:C); 2) The ISRE DNA

elements, to screen for the inhibition of the impact of type I IFN; and 3) The GAS DNA elements to screen for the inhibition of

the impact of type II IFN. These experiments revealed four ASFV genes inhibiting one or more of the three luciferase assays

and, as will be presented, targeting different intracellular signaling intermediates. Their deletion from wild type virus may

strengthen the host interferon response and so provide an attenuated form with more restricted virus spread after the initial

infection, perhaps “buying” sufficient time to allow the development of a protective adaptive immune response. The

demonstration of multiple ASFV genes for the evasion of IFN responses will demand technology to construct viruses with

multiple gene deletions. An alternative would be a multigene DNA vaccine.

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Effect of deleting African Swine Fever Virus interferon inhibitory genes on virus

pathogenesis and induction of protective responses in pigs.

Ana Luisa Reis, Lynnette C Goatley, Tamara Jabbar, Chris Netherton, Dave G Chapman, Pedro Sanchez-Cordon, Linda K Dixon

The Pirbright Institute, Surrey, UK

Keywords: type I interferon, immune evasion, pathogenesis, protection

We deleted African swine fever virus genes that encode proteins that inhibit type I interferon induction or response from the

genomes of already attenuated strain OURT88/3 or virulent strain Benin 97/1. The genes deleted included several members

of multigene families 360 and 505/530, I329L a homolog of Toll -l ike receptor 3 and DP148R. Compared to virulent isolates,

the OURT88/3 isolate has a large deletion of similar MGF 360 and 505 genes as well as interruptions of other genes, including

the EP402R and EP153R genes that encode CD2-like and C-type lectin proteins. The results showed that deletion of these

genes did not affect virus replication in macrophages but had varying effects on the induction of IFN-beta mRNA and of

interferon stimulated genes. Infection of macrophages with virulent isolates induced barely detectable levels of IFN-beta

mRNA whereas varying levels were induced by different gene deleted viruses. Deletion or interruption of multiple genes from

MGF 360 and MGF 505/530 or deletion of DP148R gene from virulent Benin 97/1 isolate resulted in virus attenuation and

induction of goof levels of protection against challenge. In contrast deletion of the DP148R or I329L genes from OURT88/3

isolate resulted in reduced protection levels compared to parental OURT88/3 virus. The resul ts indicate that deletion of

different combinations of interferon inhibitory genes offers a promising route for construction of candidate vaccine strains.

Use of a double viral gene deletion virus as a rational approach to develop ASFV live

attenuated vaccines protecting against Georgia 2007 isolate

Vivian O’Donnell1-3

, Lauren G. Holinka1, Jolene Carlson

1-2, Marialexia Alfano

1, Paul Azzarino

1, Peter W. Krug

1, Guillermo R.

Risatti2, Douglas P. Gladue

1, and Manuel V. Borca

1

1Agricultural Research Service, U.S. Department of Agriculture, Plum Island Animal Disease Center, Greenport, NY 11944, USA. 2Biosecurity

Research Institute and Department of Diagnostic Medicine and Pathobiology, College of Veterinary Medicine, Kansas State University,

Manhattan, KS 66506, USA. 3 Department of Pathobiology, University of Connecticut, Storrs, CT 06269, USA.

Keywords: experimental vaccine, recombinant virus, protection, ASFV Georgia2007 isolate

ASFV vaccine strains based in the use of recombinant viruses harboring only a single viral gene deletion that is critical for

virus virulence have been under development by us in previous studies (JVirol 2015 89:6048; JVirol 2015 89:8556). These

experimental vaccines harbor a single deletion where either the 9GL gene or six genes belonging to the MGF 360 and 505

families are deleted. Although these two virus strains were effective in inducing protection against the homologous virulent

parental virus (Georgia2007 isolate) their use with one deletion may have some potential safety issues. In order to attempt to

further attenuate our vaccines and increase the safety of a potential vaccine, we have developed two new recombinant

ASFVs derived from the Georgia2007 isolate where additional genes have been deleted in our original experimental vaccines.

We are reporting here the methodological development of those new mutant viruses that contain deletions in two different

areas of the Georgia2007 isolate, and the results obtained using these new double gene deleted viruses in terms of their

immunogenicity, and by using them as experimental vaccines that could protect against virulent isolate Georgia2007.

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Heterologous prime-boost vaccine strategy for ASF

Yolanda Revilla1, SunYoung Sunwoo

2, Lina Mur

2, Daniel Madden

2, Nick Haley

2, Igor Morozov

2, Tammy Koopman

2, Elena G.

Sánchez1, Daniel Pérez Núñez

1, Marisa Nogal

1, Carmina Gallardo

3, Marisa Arias

3, Juergen Richt

2

1Ceezad, KSU. Manhattan, KS, US -

2CBMSO-CSIC-UAM. Madrid, Spain

Keywords: ASFV, protein, DNA vaccine, heterologous prime-boost

Although immune responses against African Swine Fever Virus (ASFV) are not fully understood, previous studies suggest the

need to activate humoral and cellular immunity to obtain a protective vaccine for ASFV. Therefore, the objective of the

present study was to develop protection using a new approach based on heterologous prime-boost vaccination combining

ASFV antigens encoded by DNA plasmids and recombinant ASFV proteins. For that purpose, six proteins of ASFV were

expressed in Baculovirus (Sf9 cells) and six ASFV genes were cloned into pcDNA 3.1 expression plasmids. The immunogenicity

of the constructs were tested in two independent experiments. In total, 72 piglets were immunized with 15 different

combinations of proteins and DNA plasmids. Serum and blood samples from vaccinated animals were evaluated by ELISA,

ELISPOT and virus neutralization assays. The results identified some promising combinations of plasmid DNAs and antigens.

ELISA antibody titers were high with a number of proteins including p15, CD2-V, p54 and p35. Interestingly, the combination

of when combined of these proteins with plasmid DNA, showed increased levels of virus neutralization capacity. Specifically,

neutralizing antibodies were more efficiently induced in pigs immunized with combinations of ASFV proteins and

heterologous plasmid DNAs (i.e. p15 protein and CD2v DNA). Some of these combinations reached up to 80% of ASFV

infection inhibition levels. The results from this work opens a new avenue for future studies on the most appropriate

combination of ASFV-specific cDNAs and proteins to develop a rationally designed, safe, efficacious and DIVA-compatible

vaccine for ASF.


Paula López-Monteagudo1, Anna Lacasta

1, Andreas Gallei

2 , Veljko Nikolin

2, Javier M Rodríguez

3, Elisabet López

1, Sonia Pina

1,

Francesc Acensi1,4

, María Jesús Navas1, Laia Bosch

1,2, María Luisa Salas*

3, Fernando Rodríguez*

1

1CReSA-IRTA Barcelona (Spain) - 2Boehringer Ingelheim Gmbh Hannover (Germany) - 3CBMSO-CSIC Madrid (Spain) - 4UAB Barcelona Spain -*

Both authors contributed equally

Keywords: ASFV, Vaccine, Crossprotection, CD8 T-cells, residual virulence

The 3rd GARA scientific meeting represents the ideal forum to discuss the future of ASFV vaccinology. Conversely to the most

skeptical ones, we really believe that obtaining a safe and efficient vaccine against ASFV is not only feasible but also

necessary if will ing to control the disease, at least in endemic areas where stamping out policies are not feasible. In contrast

with the recurrent failure of classical inactivated vaccines, l ive attenuated viruses (LAV) have demonstrated to confer solid

protection against experimental homologous challenges, showing limited protection, if any, against heterologous ASFV

challenges. Here we will present some of the most recent advances obtained in collaboration between our laboratory at

CReSA-IRTA, the laboratory of Dr. ML Salas at the CBMSO-CSIC and Boehringer Ingelheim Veterinary Research Center

(Hannover, Germany). Our results demonstrated the feasibil ity of obtaining a recombinant l ive attenuated virus by gene

targeting, capable to confer protection not only against the lethal homologous virus challenge (BA71; Genotype I), but also

against heterologous virus strains from the same (E75) and from different genotypes, including Geogia07 (Genotype II), the

virus currently circulating in continental Europe. Additional evidences regarding the mechanisms involved in the

crossprotection conferred will be also presented. We will finish our presentation by discussing the risk of this and any othe r

ASFV-LAVs, mainly due to their potential residual virulence. Even if obtaining safe, efficient and affordable crossprotective

vaccines: Could l ive-attenuated African swine fever viruses become a vaccine reality? We hope that our talk will incentivize a

multidisciplinary discussion between the present experts.

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Chairs:

Dolores GRAVIER-WIDEN, Sweden Willie LOEFFEN, Netherlands

- 27

The African Swine Fever sylvatic cycle: using molecules and maths to unravel the role of

the Ornithodoros tick vector

Armanda D.S. Bastos1, Roumen Anguelov

2, Magali Jacquier

1, Carin Boshoff

1,3,4, Livio Heath

4

1Mammal Research Institute, Department of Zoology & Entomology, University of Pretoria, South Africa -2Department of Mathematics, University of Pretoria, South Africa - 3Department of Veterinary Technology, Tshwane University of Technology, South Africa -

4Transboundary Animal Disease Programme, ARC-Onderstepoort Veterinary Institute, South Africa

Keywords: Ornithodoros, PCR, phylogenetics, seasonal variation, modeling

The Ornithodoros soft tick plays a central role in the epidemiology of African swine fever (ASF) in the sylvatic setting, and

represents a significant threat to all regions to which the disease may be introduced. This is due to the almost worldwide

distribution of Ornithodoros species, many of which are capable of maintaining and transmitting the ASF virus (ASFV) under

experimental conditions. Large-scale tick surveys conducted in the 1970s and 1980s in South Africa revealed that overall

ASFV infection rates in ticks were low across a ll populations evaluated. Although surveys conducted in the last 15 years

confirm that these low overall rates of infection remain unchanged, the virus may have disappeared from at least one

population within the southern-most distributional range of ticks previously identified as ASFV-positive. We explore the

possibil ity of virus extinction in a sylvatic setting, using mathematical models, and investigate seasonal variation in tick

infection rates using molecular approaches. A mathematical model that incor porates an extinction process was developed

which identified key parameters essential to virus perpetuation that may be useful for eradication strategies. Although there

were significant seasonal differences in adults versus nymphs and males versus female ticks in the wet and dry seasons, there

was no significant difference in ASFV infection rates between seasons. The molecular results further confirmed the patchy

distribution of the virus whilst nucleotide sequencing and phylogenetic analysis of positive s amples revealed high levels of

field heterogeneity in the Kruger National Park, South Africa for ticks sampled along a 400km longitudinal gradient.

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The 1996-2002 African Swine Fever (ASF) pandemic in West Africa: Lessons learnt

Brown A.-A.1, Penrith M.L.

2, Fasina F.O.

2 and Beltran-Alcrudo D.

1

1Animal Production and Health Division, Food and Agriculture Organization of the United Nations, Vialle delle Terme di Caracalla, 00153

Rome, Italy. - 2Department of Veterinary Tropical Diseases, Faculty of Veterinary Science, University of Pretoria, Old Soutpan Road, 0110

Onderstepoort, South Africa

Keywords: West Africa, Retrospective study, African swine fever

An ASF pandemic was observed in previously unaffected countries in West Africa during 1996-2002. It started in Côte d’Ivoire

in 1996, followed by outbreaks in Benin, Nigeria and Togo in 1997, Ghana in 1999, and eventually Burkina Faso in 2003. These

newly infected countries, which had significant and fast-growing pig populations, became ASF endemic. Only Côte d’Ivoire

(and Ghana for a brief period) managed to eradicate ASF. Despite the importance of the pandemic and the generation of a

large amount of information as a result of various activities aimed at survei llance, outbreak control or disease eradication

carried out by veterinary services and international organizations, such as FAO, throughout the region, much of the data

generated have remained archived and unpublished or only partially published. As a resu lt, the dynamics and underlying

factors driving this pandemic have not been fully elucidated or exploited. This study aimed to consolidate historical

information generated by working towards the control and eradication of ASF. This descriptive analysis entailed the

evaluation and review of archived records and reports of outbreaks, data from veterinary services, veterinary consultants

and peer-reviewed publications. The analysis of this information will allow a better understanding of the disease dynamics i n

a region infected for the first time and learning how the prevention and control interventions that were implemented

worked or failed. This will help the development of better tailored, sustainable and locally sound interventions.

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Wild boar as a ASF driver

Klaus Depner1 and Vittorio Guberti

2

1Friedrich-Loeffler-Institut, Federal Research Institute for Animal Health, Suedufer 10, 17493 Greifswald - Insel Riems, Germany

2 Istituto Superiore per la Protezione e la Ricerca Ambientale, Via Ca’ Fornacetta, 9, 40064 Ozzano Emilia, Italy

Keywords: ASF, wild boar, persistence, contagiosity

After the genotype II of ASFV reached the eastern borders of the European Union in January 2014 two main epidemiological

scenarios were forecasted. ASF would fade out spontaneously from the local wild boar population or, alternatively an

epidemic wave would start moving westward very rapidly, affecting large areas of Europe. However, after almost two and a

half years both epidemiological hypotheses proved to be wrong. The virus did not fade out nor assumed an epidemic wave

behavior. On the contrary the infection survived locally with a steady low prevalence (below 3%). Field data as well as

experimental studies on ASF indicate an overall high case-fatality rate and a rather low contagiosity and low mortality during

the initial phase of infection.

Current findings indicate that in wild boar populations ASF shows a pattern of habitat bound persistence lacking a tendency

of dynamic spatial spread. Therefore ASF in wild boar can be considered a habitat-borne disease where infected carcasses in

combination with the tenacity of the virus and the low contagiosity (contagiosity index <0.3) play a key role in capturing the

disease within particular areas. In such affected areas wild boar are becoming the drivers for ASF. Such circumstances are

l ikely to contribute substantially to months or even years of pathogen persistence explaining the current picture of ASF

spreading rather slowly and with continuing circulation in affected wild boar populations. Furthermore, the wild boar density

appears to be less important concerning virus persistence whereas the number of dead animals which are highly infectious

and which may remain in the fields for weeks until they are completely decomposed can play a more relevant role.

Within this context, a revision of the current unders tanding and approaches towards ASF control and eradication is needed.

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Krzysztof ŚMIETANKA

ASF surveillance in Poland: update on current situation

Krzysztof Śmietanka1, Andrzej Kowalczyk

2, Grzegorz Woźniakowski

2, Edyta Kozak

2, Magdalena Łyjak

2,

Małgorzata Pomorska-Mól2, Zygmunt Pejsak

2, Krzysztof Niemczuk

1Department of Epidemiology and Risk Assessment - 2National Veterinary Research Institute, Department of Swine Diseases

Keywords: African swine fever virus, polymerase cross-linking spiral reaction, pigs and wild boars

Since the 1st of January 2014 til l 30th of April 2015, 92 cases of ASF in wild boar (WB) and 3 outbreaks of ASF in domestic p igs

(DP) have been detected in Poland. These events occurred in the same area of 11 municipality in Podlaskie region. Samples of

blood and internal organs were collected. The sections of tissues were processed as 10 (w/v) homogenates in PBS then

submitted for DNA extraction. Next, real -time PCR was conducted with primers and probe complementary to the conserved

p72 gene sequence (Fernandez-Pineiro et al. 2013). ELISA was conducted using the Ingezim PPA Compac 1.1.PPA K3 ELISA kit

(Ingenasa). Immunoperoxidase test (IPT) was performed using fixed VERO infected Ba71V ASFV cells on 96 -well plates. All

samples from DM and WB were tested by real -time PCR. In total 33,317 examined WB, 148 were found as ASF-positive. From

all 148 ASF-positive WB, 128 were positive only in PCR (including 11 from hunted WB), 7 positive by PCR and serological

methods (including 3 from hunted WB), and 14 only in serological methods (all 14 from hunted WB). Prevalence of ASF in WB

was the highest in found dead animals (2.65%), indicating that ASF-cases in WB in Poland are caused by high virulent virus,

when infected animals supposed to die so quickly that immunological system is not able to produce antibodies. Number of

survivors was found lower than 1% (0.0565 % prevalence of serologically positive hunted WB) confirming /supporting the

thesis concerns a possibil ity of recovering small percentage of animal infected with high pathogenic type of ASFV.

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Chairs:

Dolores GRAVIER-WIDEN, Sweden Willie LOEFFEN, Netherlands

- 32

African Swine Fever Virus spread in Estonia: the preliminary results of epidemiological

analysis

Nurmoja Imbi; Kristian Maarja; Blome Sandra; Gallardo Carmina; Viltrop Arvo

Estonian University of Life Sciences, Institute of Veterinary Medicine and Animal Sciences, Estonia - Veterinary and Food Laboratory, Estonia

- Veterinary and Food Board, Estonia - Friedrich Loeffler Institute, Institute of Diagnostic Virology, Riems, Germany - European Union

Reference Laboratory for African Swine Fever, Centro de Investigacion en Sanidad Animal (CISA-INIA), Valdeolmos, Madrid, Spain

Keywords: African swine fever, epidemiology, wild boar, domestic pig

As a result of extensive spread of the highly virulent ASFV genotype II in Eastern Europe since 2007, the first case of the

disease in Estonia was confirmed in September 2014. The first infected dead wild boar in Estonia was found in the very south

of Estonia, 6 km from the Latvian border. Subsequently, the virus was detected in wild boar in 3 counties bordering Latvia,

over the following three weeks, but also in northeast Estonia in a location over 200 km from the other ASFV affected

counties. The epidemiology of the infection in these two areas was different during the first year of the epidemic – in the

southern outbreak area high mortality among wild boar was observed. However in the northeast outbreak area there was no

mortality, and among hunted animals most wild boar were only antibody-positive. A challenge experiment conducted at FLI

in Germany, with the virus isolated from the northeast outbreak, revea led that the virus was virulent causing 90% lethality

among challenged animals. This indicates that the incursion of the virus into northeast Estonia could have happened much

earlier. Starting from July 2015, the ASFV situation in wild boar changed drastic ally, when the virus spread fast to new

previously uninfected areas. In addition, during the eight-week period in summer 2015, 18 outbreaks in domestic pigs were

confirmed. In total, 1 836 ASFV positive wild boar have been found in 12 counties between September 2014 and April 2016.

The ASFV strain currently circulating in Estonia, belongs to the p72 genotype II, which has been circulating in Eastern

European countries since 2007. However, in July 2015, on the basis of the central variable region (CVR) -PCR product analysis,

a new genetic variant of the virus was detected in wild boar in southern Estonia.

Evaluation of insect larvae as possible mechanical vectors for transmission of ASFV in

wild boar populations

Forth JH1, Amendt J

2, Blome S

1, Depner K

1, Kampen H

1

1Friedrich-Loeffler-Institut, Federal Research Institute for Animal Health, Greifswald – Insel Riems, Germany - 2Goethe University, Institute of

Forensic Medicine, Frankfurt, Germany

Keywords: African swine fever virus, blow fly, maggots, mechanical vector

African swine fever (ASF) is a contagious viral disease of domestic pig and European wild boar. In 2007, ASF virus (ASFV) was

introduced into eastern Europe, reaching the EU in 2014 (Baltic States, Poland). Considering the rapidly progres sing course of

disease with case fatality rates of up to 100 %, it is unclear why the virus keeps slowly spreading through the East European

wild boar populations in the absence of the only known vector (Ornithodoros soft ticks) instead of quickly eradicat ing its

natural hosts and, ultimately, itself. In addition to direct contact to infected animals with high virus load, pigs and wild boar

can become infected by oral ingestion of ASFV-containing organic material in which the virus remains stable for weeks or

even months. Thus, transmission might also be possible through carcasses and insect larvae developing on those, which are

known to belong to the diet of wild boar. To check the hypothesis of immature insect stages serving as mechanical vectors of

ASFV, larvae of two commonly found necrophagous blow fly species, Lucil ia sericata and Calliphora vicina, were bred in the

laboratory on ASFV-infected wild boar tissue. After preset periods of development, maggots were removed and tested for

replicating virus via titration on porcine macrophages and for viral DNA by qPCR, both in an unwashed status and following

external cleaning. The study is meant to contribute to understanding the epidemiology of ASF in eastern Europe and to

predicting the further spread of ASFV within the wild boar population.

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Quantification of transmission of African Swine Fever Virus by carriers and through

indirect environmental contact

P.L. Eblé, E. Weesendorp, H.W.M. Moonen-Leusen, S. Quak, T.J. Hagenaars, W.L.A. Loeffen

Central Veterinary Institute of Wageningen UR (CVI), Lelystad, The Netherlands

Keywords: ASF, virus, carrier, transmission

The Eastern European ASFV strain seems almost 100% lethal. After infections with less virulent strains, however, several pigs

survive the infection and become carriers, defined as pigs recovering from the acute disease, no longer showing clinical signs,

but virus positive and potentially being infectious. The infectivity of these carriers was determined in an experimental

transmission study. Twenty pigs were inoculated with ASFV through the intranasal route with the moderately virulent strain

Netherlands ’86. Six pigs survived the acute phase of the infection and became carriers. These six carriers were housed in

clean pens, 28 days after they were inoculated, one pig per pen. In each pen, one contact animal was added for a maximum

of 13 days, or until the contact animal became infected. After 13 days, all remaining contact pigs were removed and 6 new

contact pigs were added 24 hours later for another 13 days (or until they became infected). Between day 28 and 41 after the

inoculation, none of the contacts became infected with ASFV. Between day 42 and 55 after inoculation, two of the six

contacts became infected with ASFV. The overall transmission rate in carriers was estimates to be 0.019 (0.0027-0.048) per

day. This is much lower than in the acute phase of the infection, where transmission rates for this particular virus range fr om

0.45 to 0.92 per day. Even though virus transmission from carrier s is very l imited, on a population level this could be a crucial

factor for the virus to become endemic in wild boar.

Active management of ASF spread in wild boar: strategic options and plausible

solutions Hans-Hermann Thulke, Martin Lange

Helmholtz Centre for Environmental Research Leipzig - UFZ

Keywords: wild boar, control measures, population reduction, model

African swine fever (ASF) is an emerging viral disease in Eastern European domestic pigs and wild boar. Since its introductio n

into Poland and the Baltic states in early 2014, ASF spreads continuously in wild boar populations, despite its successful

control in domestic pigs. The objective of this study was the investigation of alternative ad-hoc concepts proposed to halt ASF

spread in wild boar. For the moment vaccination of wild boar is not an option. All expert debates regarding useful control

measure target population regulation but with alternative measures and timelines. Here, the two extreme concepts are

compared in a virtual wild boar population affected by ASF: 1) a drastic, active depopulation (e.g. removal of 80% of the

population within four months), versus 2) “soft” passive population reduction through usual hunting but targeting any female

able to reproduce for multiple hunting seasons. To quantitatively compare the strategy concepts, an individual -based

epidemiological model was implemented. The model was parameterized from literature regarding the demography of wild

boar, the epidemiology of ASF, any others were calibrated to the spatio-temporal dynamics of the most recent development

of ASF in Eastern Europe. Applying the two candidate measures over different dimensions and variable effectivity lead to the

following insights: Drastic depopulation towards less than 20% of the initi al density showed a timely effect and required a

control zone of about 50 km width. Targeted hunting applied as efficient, was effective in the prevention of spread after at

least two years of application. This would require a control zone of about 200 km width. The practicability of such control

measures can be debated. Therefore, we applied the two approaches in combination in a strutured approach. The improved,

more practical dimensions of measures in space and over time are presented. The mixed strategy was useful to halt spread of

ASF in the model population. The role of carcass, resp. their removal, will be addressed in the context of these measures.

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Towards quantitative understanding of the role of carcasses in the transmission of ASF

in wild boar or feral swine Martin Lange, Hans-Hermann Thulke


Keywords: wild boar, uncertainty, transmission, carcasses

In Eastern Europe, African swine fever was found spreading predominantly in wild boar populations. However, experimental data suggest

l imited transmission of ASF vi rus (ASFV) through direct contact between living hosts. The transmission through vi rus -laden blood is wel l

acknowledged and confirms the specialisation of the vi rus to vector tra nsmission (soft ticks). In Eastern Europe the missing vector l ink but

continuous spread in wild boar populations pointed to the carcasses as possible driver in the host-to-host transmission of ASFV in wildl i fe.

Li terature as well as expert opinions are contradicting regarding the functioning of carcass mediated transmission of ASFV. The reason is

the special social behavior observed with wild boar towards dead conspecifics. The knowledge gap was the accessibility of dea d animals for

non-mate wild boar groups (between group contacts) and the approach behaviour of wi ld boar in the vicini ty of dead conspeci fics

(transmission risk given an accessible carcass). In this study we estimated the two unknowns by compi l ing observational data of ASF

noti fication in wild boar with an agent-based spatial-explicit ecological model simulating spread of the disease on the landscape sca le.

Methodologically, thousands of ASFV epidemics were simulated on the same geography where the observational data was col lected

s tarting from the reported scene of introduction. In the simulations the two uncerta in tra i ts of ASF were varied across the complete

technical range (0-1). Subsequently, vi rtual sampling and a set-theoretic vers ion of the confus ion matrix was used to identi fy those

s imulations that were congruent with the observational data. The data-driven procedure selected parameters in favor of hypotheses that

argue about the severity of the cl inical phase of the infection which would disable systematic retreat of sick animals, i.e. greater va lues of

the carcass accessibility. Truly infective contacts to carcasses , however, should be rather sporadic, i .e. minimal chance of actual

transmission. Moreover, findings definitely exclude assumptions of targeted and intensive resource use th rough scavenging. The findings

enhance comparative assessments of control measures in wild boar, stipulate adequate field experimentations and increase awareness for

the carcass i ssue.

Research on African Swine Fever Threat Reduction Through Surveillance in the Ukraine:

An Update

Serhii Fi latov1, Anton Gerilovych

1, D. Scott McVey

2, Borys Stegniy

1, Andriy Buzun

1, Vitaly Bolotin

1, Adalberto A. Pérez de León

3

1National Scientific Center Institute of Experimental and Clinical Veterinary Medicine, Kharkiv, Ukraine - 2 United States Department of Agriculture,

Agricultural Research Service, Arthropod-borne Animal Diseases Research Unit, Manhattan, Kansas 66506, U.S.A. - 3United States Department of Agriculture

– Agricultural Research Service, Knipling-Bushland U.S. Livestock Insects Research Laboratory and Veterinary Pest Genomics Center, Kerrville, Texas, U.S.A.

Keywords: African swine fever, soft ticks, epidemiology, wild boar, Ukraine

African swine fever (ASF) is a high-consequence vi ral disease that threatens the pig farming industry in Europe. ASF outbreaks have

occurred in Ukraine. The factors driving the spread of ASF among domestic swine and wild boar populati ons in the region remain to be fully

understood. Our collaborative research project was established to generate science-based knowledge that could be used to implement a

system for sustainable surveillance of ASF vi rus (ASFV) in Ukraine. Because ASFV can replicate in, and be transmitted by certa in soft tick

species, research efforts focused on minimizing the cost of early ASFV detection by determining the necessary sample size of soft ticks and

suids for accurate detection in a local region; perform small scale surveillance efforts in selected regions to establish methods for a routine,

national program; and train personnel to perform a routine, national surveillance program. ASFV infection in wild boar popula tions was

documented. The transboundary movement of ASFV-infected wild boars appears to be a risk factor for outbreaks in the Ukra ine and the

region. Soft tick surveillance was conducted in the context of global change. This is significant because the last reports of soft tick research

in Ukra ine were published more that 50 years ago. Soft tick collections evidence another ri sk factor for the emergence of ASF. Soft tick field

and laboratory research capabilities in Ukraine offer the opportunity to investigate soft tick -pig/wi ld boar-ASFV interactions through

col laboration with other science partners in the region. These efforts will help bridge the knowledge gap on the epidemiology of ASF in

Ukra ine, which is required to develop countermeasures that mitigate the burden of this high -consequence disease for the pig farming

industry in Europe. *USDA is an equal opportunity provider and employer. This research project was funded by the U.S. Defens e Threat

Reduction Agency (CBEP Agreement IAA# U.S.C. 3318(b) – 15217).

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Chairs:

Eric ETTER, South Africa Charles MASEMBE, Uganda

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Charles MASEMBE

How important are wildlife and domestic livestock interactions for African Swine Fever

endemic situation in Africa

Charles Masembe, Karl Stahl

Makerere University, College of Natural SciencesDepartment of Zoology, Entomology and Fisheries Sciences Box 7062, KAMPALA, U ganda

Keywords: African swine fever, bushpig, livestock, warthog, wildlife

The pig industry in many sub-Saharan African (SSA) countries is on the rise and is anticipated to continue growing in the coming

years. This growth is mostly happening in developing countries and 75% of this is practiced by smallholder farmers. The popu lation

of pigs in SSA has increased fourfold in the last 50 years. This industry is however faced by the lethal and devastating African swine

fever (ASF) disease. ASF exists both in the wild and the domestic pigs. There is a high chance of ASF transmission between wild and

domestic pigs due to the porous interface. The main wild species of concern are the warthog (Phacochoerus africanus), the bushpig

(Potamochoerus larvatus), and the soft tick (Ornithodoros genus). in order to appreciate the role played by the wildlife in t he

maintanace, and transmission of ASF, concerted efforts are needed to investigate the existence, prevalence, and characteristics of

the ASF virus from each of the above species. This keynote presentation will highlight the potential role played by the key s pecies

and point the challenges faced while conducting research on ASF in Africa.

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Epidemiology of African Swine Fever in Africa today: Sylvatic cycle versus socio-

economic imperatives – filling the gaps

Penrith Mary-Louise1; Bastos Armanda D.S.

2; Etter Eric

3,4; Beltran Alcrudo Daniel

5

1Department of Veterinary Tropical Diseases, University of Pretoria; 2Mammal Research Institute, University of Pretoria; 3CIRAD, AGIRs Research Unit, 34398

Montpellier, France; 4Department of Production Animal Studies, University of Pretoria; 5Animal Production and Health Unit, Food & Agriculture Organization

of the United Nations, Rome

Keywords: ASF; warthog; domestic pig; outbreaks

The diagnosis of African swine (ASF) fever in Kenya in 1910 and its association with wild African suids (warthogs and to a lesser

extent bush pigs) and the well -documented sylvatic cycle between warthogs and soft ticks of the Ornithodoros moubata complex

sharing their burrows has resulted in an indelible impression that wild suids are the major player in ASF transmis sion in Africa.

Available information about more than 4,900 outbreaks reported to the World Organization for Animal Health (OIE) by 33

countries in Africa in the period 1996 – 2016 has been examined. The sylvatic cycle (SC) involving warthogs is known to occur in

10 of the countries and suspected but not confirmed in 5 more (Angola, Burundi, Congo Republic, Democratic Republic of Congo

and Rwanda). These SC countries accounted for 48.9% of the reported outbreaks. Therefore 51.1% of the outbreaks occurred in

15 countries where wild suid involvement could be excluded. The probable cause of 61.1% of all reported outbreaks in SC

countries could not be determined from the available information. In three SC countries (Botswana, Namibia and South Africa)

90 to 100% of the outbreaks originated from warthogs/ticks. However, in the remaining SC countries, warthog involvement was

suspected in on ly 7 outbreaks (6 in Zambia and 1 in Zimbabwe) (0.7%), with 939 (99.3%) outbreaks reported to have originated

within domestic p ig populations, predominantly as a result of movement of live pigs, feeding of uncooked swill containing

infected pork, and scav enging by free-ranging pigs. The predominance of primary domestic outbreaks makes investigation of the

socio-economic issues driving risk practices in pig husbandry and trade (already identified in several countries) imperative for

better management of ASF.

Quantitative assessment of socio-economic impacts of African Swine Fever outbreaks

in northern Uganda

Erika Chenais1,2

, Susanna Sternberg-Lewerin2, Sofia Boqvist

2, Ulf Emanuelson,

Tonny Aliro3, Emily Ouma

4, Karl Ståhl

1,2

1National Veterinary Institute, Uppsala, Sweden - 2Swedish University of Agricultural Sciences, Uppsala, Sweden - 3 Directorate of Production

and Marketing, Gulu District Local Government, Uganda - 4International Research Institute, Kampala, Uganda

Keywords: smallholders, social impacts, economic impacts, ASF outbreaks

Outbreaks of infectious animal diseases may disrupt the livelihoods of poor people in the same ways as civil unrest or other

catastrophes. In poor households these impacts are aggravated as the animals represent multiple roles. Uganda is a low-income

country with the largest pig population in east Africa. The majority of pigs are kept by smallholders. African swine fever (ASF) is

endemic in the domestic pig population. The objective of this study was to investigate the socio-economic impacts of ASF outbreaks

in northern Uganda in a longitudinal survey. Structured interviews with two hundred, randomly selected, pig-keeping households

were undertaken three times with six months interval. Questions related to family- and pig herd demographics, social and economic

variables, pig trade and pig business. The study showed that pigs were kept in extreme low-input-low-output farming systems

involving very small monetary investments. Household ASF yearly incidence was nineteen percent. Outbreaks resulted in lower

gross margin income from the pig production. This negative effect could partly be explained by the households’ poverty status and

the size of the pig herd. Trade and consumption of sick and dead pigs were coping strategies used to minimize losses of capital and

animal protein. The results indicate that causality of economic impact in smallholder systems is complex. Pigs are mostly kept as

passive piggy banks rather than active working capital, disqualifying disease control arguments based only on standard economic

models and instead requiring solutions adopted to the local situation. The quantifications of the losses incurred by ASF outbreaks

can serve to advice international bodies and governments towards the value of improved animal health in low-income countries.

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A molecular and ecological investigation of the role of the bushpig in the epidemiology

of African Swine Fever at the wildlife-livestock interface in Uganda

P. Ogweng1, T. Aliro

2, J.F. Mayega

1, D. Muhangi

1, K. Sahl

3,4 and C. Masembe

1

1 Makerere University, Kampala, Uganda - 2Gulu District Local Government, Gulu, Uganda -

3National Veterinary Institute, Uppsala, Sweden -

4 Swedish University of Agricultural Sciences, Uppsala, Sweden

Keywords: African swine fever, bushpig, Potamochoerus larvatus, epidemiology, and wildlife-livestock interface

African swine fever is a devastating hemorrhaging fever of pigs, caused by the Africa swine fever virus (ASFV). The disease

causes up to 100% mortality in domestic swine, depending on the strain. The bushpig, Potamochoerus larvatus, is known to

be susceptible to ASF and yet it shares a natural interface with both warthogs and domestic pigs. The bushpig’s role in the

epidemiology of ASFV is not adequately understood. This study used a molecular and ecological approach to investigate the

role of the bushpig in the epidemiology of ASFV at the wildlife-livestock interface in Uganda. GPS/GSM tracking collars were

used to monitor the movement pattern of free-ranging domestic pigs and bushpigs. Five free-ranging domestic pigs at the

wildlife-livestock interface were collared for two weeks. Three bushpigs were captured and collared for two months. ASFV

detection was done in domestic and wild pig samples using RT-PCR. A new-recorded home range of 8.5 square kilometers of

bushpigs in Uganda was observed. Interestingly, there was an overlap between free-ranging domestic pigs and bushpig

activity times, indicating the possibil ity of interaction between the two species. 14 free-ranging domestic pig blood samples,

and two bushpig blood samples tested positive for ASFV. The bushpigs could be playing a role in ASF epidemiology at the

wildlife-livestock interface and serve as a l i nk between the sylvatic and non-sylvatic cycle.

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Experimental versus natural determinants influencing the vector competence of

Ornithodoros soft ticks for African Swine Fever Virus

Bernard J.1, Madeira S.

2, Boinas F.

2, Vial L.

1

1CIRAD, UMR CMAEE, F-34398 Montpellier, France - 2FMV-UTL, Departamento de Sanidade Animal, Avenida da UniversidadeTécnica, 1300 -477

Lisboa, Portugal

Keywords: Ornithodoros soft ticks, African swine fever virus, vector competence, determinants

African Swine Fever (ASF), presently occurring in Africa, Caucasus and Eastern Europe, is one of the most economically

devastating viral diseases for swine. Although ASF is contagious, soft ticks of the genus Ornithodoros can be involved in the

epidemiology of the disease in some countries, as vectors and natural reservoirs of African Swine Fever Virus (ASFV). As soon

as ASFV is introduced in a free area where the presence of Ornithodoros soft ticks is confirmed or suspected, one main issue

is the possibil ity of ASFV transmission through tick bites and the long-term persistence of ASFV in tick reservoirs because of

the obvious difficulties to eradicate such ticks in pig buildings. The vector competence of soft ticks for ASFV has been main ly

studied by mimicking natural tick-to-pig and tick-to-tick transmission cycles through experimental infections in laboratory. As

a consequence, experimental design can highly impact the vector competence results, as far as experiments may be

sometimes limited by technical inherent constraints. By selecting and reviewing the 32 available papers and theses published

since 1969 to date on the vector competence of Ornithodoros soft ticks for ASFV, it was possible to identify the different

ways to define vector competence depending on authors , and to highlight the diversity of methodologies used to test and

measure ASFV vector competence. An analysis of experimental (tick infection route, ASFV titer in blood meal, tick colony

status and tick development stage…) versus natural (tick species, ASFV isolate, host of ASFV isolate…) factors that may

influence tick infection with ASFV was helpful to propose optimized processes to quickly and efficiently test the vector

competence of Ornithodoros soft ticks for ASFV. Knowledge of the potential biases d ue to the methodology is also useful to

be much more critical on results and be able to conclude on general patterns of ASFV vector competence.

The potential role of the bushpig (Potamochoerus larvatus) in the spread and

maintenance of African Swine Fever at the wildlife-livestock interface in Uganda

PAYNE Ariane1, OGWENG Peter

1, JORI Ferran

2,3, RASMUSSEN Henrik Barner

4, MASEMBE Charles

1, STAHL Karl

5

1Makerere University, College of Natural Sciences, Department of of Zoology, Entomology and Fisheries Sciences, P.O. box 7062, Kampala, Uganda. - 2CIRAD, Integrated Animal Risk Management Unit (AGIR), Montpellier, France - 3Botswana University of Agriculture and Natural Resources (BUAN),

Department of Animal Science and Production, Sebele, Botswana - 4Savannah Tracking Ltd, Nairobi, Kenya - 5National Veterinary Institute,

Department of Disease control and Epidemiology, Uppsala, Sweden

Keywords: African swine fever, Potamochoerus larvatus, wildlife-livestock interface, Uganda

In Uganda, African swine fever (ASF) is endemic in domestic pigs and causes large economic losses for rural communities.

Although warthogs (Phacochoerus africanus), in association with soft ticks are considered the main wild vertebrate host of

the virus in the endemic African setting, they are not the only wild African suids with a potential role in ASF epidemiology.

The sylvatic cycle also involves the bushpig (Potamochoerus larvatus) but to date, l ittle is known about the epidemiological

role of this species in the maintenance and spread of ASF, although it is l ikely to share habitats and resources with both,

warthogs and free ranging domestic pigs. A previous pilot study carried out in Uganda showed that bushpigs can be naturally

infected with ASF virus and can seroconvert. The aim of this ongoing study is to increase the sample size of captured and

sampled bushpigs 1) to be able to estimate ASF prevalence and seroprevalence and 2) to better evaluate their temporal and

spatial interaction pattern with domestic pigs. Blood from bushpigs has been sampled and tested for ASF virus genetic

material and antibody detection. We have fitted domestic pigs and bushpigs with GPS harnesses, estimate th eir home ranges

and use of habitat to determine where and when the interspecies contact is more likely to occur. The expected results should

highlight the role of the bushpig in the sylvatic cycle of ASF and the way it could be connected with the domestic cycle.

Further research will then be needed to investigate what are the route of transmission and if this species is able to mainta in

the virus.

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Integrated study of African Swine Fever in East Africa identify the disease drivers and

provides insights for targeted surveillance and control

Edward Okoth-Abworo1, Jocelyn Davies

2, Cynthia Onzere

1, Joshua Amimo

7, Jacqui Kasiiti Lichoti

3, Selestine Naliaka

7, Beatrice

Abutto3, Yiheyis Maru

2, Sam Okuthe

4, Patrick Barasa

5, Noelina Nantima

5, Joel Manyonge

6, Richard P. Bishop

1

1International Livestock Research Institute and Biosciences East and Central Africa - 2CSIRO Ecosystem Sciences - 3Kenya Ministry for

Agriculture, Livestock and Fisheries & The University of Nairobi - 4FAO Emergency Centre for Trans-boundary Animal Diseases Operations

(ECTAD) - 5Ministry of Agriculture, Animal Industry and Fisheries - 6Busia County Government, Kenya - 7University of Nairobi

Keywords: African swine fever, Epidemiology, Maintenance ,Transmission, Control

We conducted studies to generate insights into factors influencing African swine fever (ASF) occurrence and control.

Epidemiological research linked to laboratory diagnostics, mathematical modeling, social networks study and value chain

analyses were used to generate evidence for control. ASF virus in internal tissues of healthy pigs demonstrated a carrier

state. A 17% prevalence of exposure of pigs to bites by the ASF virus tick vector (Ornithodoros Moubata) was shown. High

burden of other super-infections including helminthes, enteric viruses and ectoparasites was demonstrated in the pigs,

portending reduced to increased host resil ience and thus susceptibil ity to ASF virus. Critical ASF transmission paths and

nodes using social networks analysis showed that pig value chain actors play a critical role in spreading the disease.

Molecular characterization of ASF viruses in the area showed close similarity and limited diversity consistent with historica l

East African viruses from a genotype IX family. The study identified drivers for virus maintenance, transmission and spread,

this understanding not only important in tracking virus movement but also important for trans -boundary surveillance of ASF.

Results suggest need for innovative approaches for Prevention, Detection and Response (P DR) to ASF based on the

epidemiological evidence to mitigate transmission and spread locally and globally.

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Chairs:

Carmina GALLARDO, Spain Armanda BASTOS, South Africa

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Molecular characterization of Sardinian African Swine Fever Viruses based on analysis

of p30, CD2V and I73R/I329L variable region

Dei Giudici Silvia1, Sanna Giovanna

1, Franzoni Giulia

1, Bacciu Donatella

1, Bandino Ennio

1, Liciardi Manuel

1, Ruiu Angelo

1,

Giammarioli Monica2, De Mia Gian Mario

2, Oggiano Annalisa

1

1Istituto Zooprofilattico Sperimentale della Sardegna, via Duca degli Abruzzi 8, 07100 Sassari - 2Istituto Zooprofilattico Sperimentale

dell’Umbria e Marche, via Salvemini 1, 06126 Perugia

Keywords: African swine fever virus genotyping; Sardinia; CD2v

African Swine Fever (ASF) is an highly lethal hemorrhagic disease affecting pigs. ASF is present in Africa, in several Easter n and

Central European countries and in Sardinia (Italy). Previous s equencing data of genes that codify for viral protein p54, p72

and the Central Variable Region (CVR) within the B602L gene, revealed that Sardinian isolates show a very low level of

variability. In this study we have chosen three different genome regions to investigate the within-genotype relationships and

to provide a more accurate assessment of the origin of outbreaks. The analysis of p30 and I73R/I329L sequences obtained

from ASFV collected in Sardinia over a 13–year period confirms a remarkable genetic stability. The sequence comparison of

the protein encoded by the EP402R gene (CD2v), carried out on various strains from 1978 to 2014, revealed a temporal sub -

division of Sardinian viruses into two sub-groups: one group includes isolates from 1978-1990 and the second one is

comprised of the viruses collected in the period 1990- 2014. These data, together with those obtained from CVR within the

B602L gene analysis, demonstrated that the viruses circulating in Sardinia belong to p72 genotype I, but have under gone

genetic variations in two different regions of the genome since 1990. We proposed the cytoplasmic region of CD2v protein as

a new genetic marker that could be use to analyze ASFVs from different locations in order to track virus spread. Our study

reaffirms the need to analyze other genome regions in order to improve the molecular characterization of ASFV.

African Swine Fever (ASF) genotype Il in Zimbabwe durina 2015

Juanita van Heerden1, Biko M. Gadaga

2, Reverend M. Spargo

3, Kerstin Malan

1

1Agricultural Research Councii-Onderstepoort Veterinary lnstitute, Transboundary Animal Diseases, Onderstepoort, South Africa. - 2Division

of Veterinary Services, Diagnostics and Research Branch, Central Veterinary Laboratory, Harare, Zimbabwe. – 3Division of Veterinary

Services, Epidemiology and Disease Control Branch, Causeway, Harare, Zimbabwe

Keywords: Genotype, Zimbabwe, domestic pigs

Zimbabwe borders South Africa to the south, Botswana to the west, Zambia to the northwest, and Mozambique to the east

and northeast. An outbreak of a severe haemorrhagic disease in domestic pigs based on clinicat signs was reported to

veterinary officiais in July 2015 in the Mashonaland Central area, Zimbabwe. During the 2015 suspected outbreak of ASF the

first cases were reported in Foya and Mukumbura, Mt Darwin, Mashonaland Central, Zimbabwe on 10 July 2015 in free-

ranglng swine of varying age groups. The aim of this study was to characterise the African swine fever viruses (ASFV)

responsible for the 2015 outbreak in Zimbabwe. The infection, post mortem and laboratory reports confirmed the incidence

of ASF. Molecular diagnosis and characterisation of ASFV outbreak stralns was performed on tissue samples collected in July

2015 from domestic pigs. Genetic characterisation of the variable 3'-end of the B646L gene and complete E183L gene as weil

as the central variable region (CVR) of the 9RL ORF indicated that the 2015 outbreak of ASF viruses grouped with other

genotype Il viruses which caused outbreaks in Malawi, Tanzania, Mauritius as weil as Mozambique. The p72 and p54 data

suggest that the disease was caused by closely related ASFVs, since they had 100% nucleotide similarity. This study

constitutes the first description of genotype Il in Zimbabwe.

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Detection of antibodies to Ornithodoros saliva antigen in domestic pigs from Cross

River and Taraba states, Nigeria: Pilot Study

Luka PD, Mwiine FN, Yakubu B, Perez-Sanchez R, Shamaki D, Unger H

Biotechnology Division, National Veterinary Research Institute, Vom, Nigeria College of Veterinary Medicine, Animal Resources and

Biosecurity, Makerere University, Kampala Uganda Parasitologia IRNASA (CSIC) Cordel de Merinas Salamanca Spain

Keywords: African swine fever, Tick Salivary antigen, ELISA, Nigeria

African swine fever (ASF) is a viral disease with severe consequences and its growing distribution is a threat to the pig

industry glabally. It is caused by ASF virus (ASFV), the only DNA virus transmitted by an arthopod vector, soft ticks of the

genus Ornithodoros. These ticks have been reported in Europe, East and Southern Africa to be part of ASFV transmissio cycle

and are responsible for genetic variations and long-term maintenance of the virus in those regions. Since 1996, ASF has

continually been reported among domestic pigs in Nigeria with a few reports from wild pigs. In an attempt to assess the risk

of tick-domestic pig transmission cycle in Nigeria, 728 serum samples were collected from domestic pigs in two states of

Nigeria (Cross River and Taraba) and analysed for anti -tick antibodies using the recombinant rtTSGP1 indirect ELISA. Of the

samples analysed, 27 showed moderate to high reactivity, which was indicative of tick bites among the sampled domestic

pigs. Positive sera were found in the two sampled states, both of them havi ng optimum climatic conditions for soft ticks as

well as national parks harboring wild swine (warthogs) able to maintain the developmental cycle of the ticks. Although this

study did not include the direct searching of soft ticks within the study area, it provides solid evidence of interaction between

soft ticks and domestic pigs in Nigeria's ecosystem. This finding will support further studies to establish the role of tick s in

the epidemiology of ASF and informed control and eradication in Nigeria.

Epidemiological study and socio-economic impact associated with persistence of African swine fever in Southern highlands of Tanzania.

Misnzo Gerald1, Yona Clara

2

1

Sokoine University of Agriculture, Morogoro, Tanzania - 2Department of microbiology and parasitology - Sokoine university of agriculture,

Morogoro, Tanzania

Keywords: African swine fever, Northern and Southern Tanzania, Genetic variability, Epidemiology, Genome sequencing,

persistence factors, next generation sequencing

In Sub-Saharan Africa, African swine fever is the disease that has caught attention since it is bringing great loss in the Agriculture

field and to the society, and Tanzania is among the countries that are most affected by this disease. ASFV is maintained in a sylvatic

cycle between wild swine (warthogs and bushpigs) and argasid ticks of the genus Ornithodoros. Deaths of pigs especially in the

places with high pigs’ population have been experienced in the Northern and Southern parts of Tanzania where most pigs are kept

due to the frequent outbreaks of African swine fever. The genetic nature of the ASF viruses involved in the outbreaks has been

partially determined and has indicated persistent circulation of ASF within the pig population rather than its emergence from

warthogs. The main objective of my research is to investigate the genetic variability and relatedness of ASFV and factors that lead to

persistence of ASF in Tanzania. The specific objectives are to investigate the epidemiology of ASF through genome sequencing of

ASFV recovered from pigs during new outbreaks and in archived samples, to investigate the factors that lead to persistence of ASF

in order to enhance the implementation of simple ASF control approaches and to determine the socioeconomic impacts of ASF, an d

lastly to provide education to the farmers upon the implantation of simple ASF control approaches. The methodologies in this

research are developed respectively to the objectives outlined; epidemiological l inks between different ASF outbreaks will be

determined by comparing ASFV recovered from pigs, warthogs and ticks, genome sequencing of ASFV in collected and archived

samples will be done using a combination of dideoxynucleotide and next generation sequencing.. For the socioeconomic impact and

control options for ASF there will be the collection and analysis of quantitative a nd qualitative data to assess knowledge. The main

outcome that is expected from my study is to bring about the total control of African swine fever virus in Tanzania and to help the

African society all together in searching for better l ives of people especially farmers without the presence of ASF. This study will

help with the controlling of African swine fever in Tanzania that has brought about high loss and low socioeconomic status in the

society.

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A new African Swine Fever Virus strain of genotype I circulates among pig herds in

southwest Nigeria

Emmanuel Jolaoluwa Awosanya1, Babasola Olugasa

1, Gimba Fufa

2, Farouk Bande

3, Yusoff Sabri

2

1Department of Veterinary Public Health and Preventive Medicine, Faculty of Veterinary Medicine, University of Ibadan, Ibadan, Nigeria - 2Department of Veterinary Pathology and Microbiology, Faculty of Veterinary Medicine, Universiti Putra Malaysia, 43400 U PM, Serdang, Selangor,

Malaysia - 3Department of Virology, Faculty of Veterinary Medicine, Universiti Putra Malaysia, 43400 UPM, Serdang, Selangor, Malaysia

Keywords: African swine fever virus, sequencing, phylogeny, Southwest Nigeria

Twenty two different genotypes of African swine fever (ASF) virus based on the p72 gene circulate on the African continent. Nigeria

had experienced epizootics of ASF in pig herds between 1997 and 2005 in the southwest region of the country; with the implicating

strains identified. However, ASF is believed to be currently enzootic in southwest Nigeria; and the circulating ASF virus strain in the

enzootic state is unknown. This study aimed at identifying the current circulating field strain (s) of ASF virus and the evol utionary

trend overtime in southwest Nigeria. DNA extraction of 144 pooled blood samples by farms was done according to the

manufacturer’s description. The extracted nucleic acid was amplified using the conventional PCR targeting p72 gene with expec ted

band weight of 850bp. The amplified PCR products were sequenced by Sanger sequencing and the genomic sequence analyzed

using BioEdit, BLAST and MEGA 6 software. Of the 144 pooled blood samples by farms, only 11 (7.6%) had bands at 950bp. A new

field strain of ASF virus of genotype I that shared the same ancestry with ASF virus strains or isolates from Spain and Brazil was

identified as circulating among pig herds in the enzootic state. The new field strain also differs in amino acids composition from

previously identified ASF virus field strains in southwest Nigeria during epizootics. The current circulating field strain of ASF virus

identified is suggestive of a mutational change likely responsible for the decrease in morbidity and mortality recorded in sp oradic

cases.

Rapid detection and epidemiological surveillance of African Swine Fever using oral fluid

David T. Will iams1, Andrea Certoma

1, Brenton Rowe

1, Dayna Johnson

1, Luis G. Gimenez-Lirola

2, Jeffrey J. Zimmerman

2,

Alfonso Clavijo3

1CSIRO, Australian Animal Health Laboratory, Geelong, Australia. - 2Department of Veterinary Diagnostic and Production Animal Medicine,

College of Veterinary Medicine, Iowa State University, Ames, Iowa USA. - 3Department of Diagnostic Medicine and Pathobiology, Kansas

State University, Manhattan, Kansas USA.

Keywords: Diagnostics, oral fluids, surveillance

In the event of an African swine fever virus (ASFV) outbreak, the key to rapid detection and control will be immediate access to

accurate diagnostic tools and an efficient surveillance (sampling) strategy. The major barrier to efficient surveillance is the

complexity and cost of collecting and testing statistically appropriate numbers of samples. Oral fluid-based diagnostics provide a

possible solution by allowing pen-based sampling. Using specimens derived from an experimental ASFV contact-infection model, we

compared the diagnostic performance of a selection of commercially available diagnostic kits and OIE-recommended tests for the

detection of specific antibody and viral DNA. A comparison of diagnostic performance as a function of specimen was also performed

(i.e., oral fluid vs. serum vs. fecal specimens). In three groups of pigs (n=6, n=8, n=16), 2, 2 and 4 "donor" pigs, respecti vely, were

intramuscularly inoculated with the moderately pathogenic Malta/78 strain of ASFV and then returned to the remaining (contact)

pigs in each group. Donor pigs developed severe disease between 6 and 11 dpi and were euthanased having reached

predetermined humane endpoints. Contact pigs began showing clinical signs from day 5 dpi, which progressed to severe clinical

signs between 14-35 dpi, at which point the majority of pigs were euthanased. Oral fluids collected with cotton rope, oral swabs,

and faeces (pen) were col lected daily and blood was taken every 2-3 days. Preliminary antibody results indicated that oral fluid

samples were comparable with individual oral swabs (100% agreement) and were consistent with the development of serum

antibodies, albeit at much lower titres. Similarly, PCR detection of viral DNA in oral fluid samples showed high levels of agreement

with individual oral swabs (88%) and whole blood samples (67%). Viral DNA was not detected in any of the pen fecal samples

tested, indicating that this sample type was not suitable for PCR testing. The results of additional testing, including comparison of

diagnostic kits, and the implications for ASFV laboratory diagnosis will be discussed in this presentation .

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Evaluation of a portable realtime PCR platform (TCOR-8) for ASF during outbreaks in an

endemically infected population in Uganda.

Liu L1, LeBlanc N

2, Atim S

3, Esau M

3, Chenais E

1, Mwebe R

3, Nantima N

3, Okurut RA

3, Ayebazibwe C

3 and Ståhl K

1

1National Veterinary Institute, Uppsala, Sweden; 2Consultant molecular diagnostics, Uppsala, Sweden; 3National Animal Disease Diagnosis

and Epidemiology Center, Entebbe, Uganda

Keywords: African swine fever, on-site diagnosis, molecular diagnostics

The objective of this study was to evaluate a diagnostic system for the detection of ASFV on-site or in simple lab settings in

remote areas. The study was carried out within an OIE twinning project between the Swedish National Veterinary Institute,

and the Ugandan National Animal Disease Diagnosis and Epidemiology Center. Blood and tissue samples were collected from

56 sickly or apparently healthy domestic pigs during outbreak investigations in Uganda. Sample preparations included simple

dilution in PBS or nucleic acid extraction using magnetic beads -based method. A dried-down ASFV PCR kit with internal

control (Tetracore ASFV, Tetracore Inc., Rockville, Maryland) was used on a portable real -time PCR thermocycler TCOR8

(Tetracore Inc., Rockville, Maryland), performed on-site in the affected vil lages and/or in a si mple lab setting. As a reference,

the OIE recommended UPL assay was performed on a stationary instrument at NADDEC. Twenty-two samples were initially

found positive for ASFV using the Tetracore ASF kit on the TCOR8. Two samples had high Ct values (38 and 3 9.9, respectively)

and became negative when retested by the same assay, and where considered false positives. Sixteen samples were positive

regardless of sample preparation methods. Contamination occurred in one diluted sample, as the nucleic acids extract ed

from the blood sample were negative for ASFV. For the non-extracted samples, inhibition was more evident in most 20-fold

diluted compared to the 40-fold diluted blood samples, as revealed by shifting of Ct values of the IC. The results of the

Tetracore ASF on TCOR8 were in very good accordance with those of the reference method. The results have important

implications for molecular detection of ASFV on-site or in a simple lab setting. By testing non-extracted, simply diluted, blood

samples, confirmation of an outbreak can be performed within 1.5-2 hours, and appropriate actions can be taken. The

experience of performing the real -time PCR assays on-site highlighted critical factors that need to be considered, including

biosafety issues, simplicity of sampl e preparation and turn-around time.

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P01

Development of monoclonal antibodies against ASFV major structural proteins: p72, p54, and p30

Maria V. Murgia1, Vlad Petrovan

1, Fangfeng Yuan

1, Pengcheng Shang

1, Ping Wu

2, Andre Lowe

2, Mallory Phillips

1, Wei Jia

2, Ying

Fang1, Raymond R. R. Rowland

1

1Diagnostic Medicine and Pathobiology, College of Veterinary Medicine, Kansas State University, 1800 Denison Avenue, Manhattan, Kansas

66506, USA - 2Foreign Animal Disease Diagnostic Laboratory, National Veterinary Services Laboratories, Animal and Plant Health Inspection

Services, United States Department of Agriculture, Plum Island Animal Disease Center, New York, NY 11944, USA

Keywords: African swine fever virus, p72, p30, p54, monoclonal antibodies

African swine fever virus is the etiologic agent of one of the most devastating diseases of swine for which there is currentl y

no vaccine. The aim of our study was the development of monoclonal antibodies (mAbs) against three major ASFV structural

proteins (p72, p54, and p30), and their epitope mapping. The final goal is their use as reagents in the development of

diagnostic assays and research reagents. Anti -p72 and anti -p54 mAbs were produced at PIADC against the respective

proteins based on ASFV Georgia/07 strain, while anti -p30 mAbs were produced at KSU against a p30 protein based on ASFV

BA71V strain. The epitope mapping was conducted by indirect ELISA and western blotting using overlapping fragments

expressed in E.coli as antigens. A total of 29, 12, and 3 mAbs were produced for p72, p54, and p30, respectively. The p72

mAbs reacted with the region between amino acid (aa) 110 and 171, 180 and 250, 280 and 303; 5 out of 12 p54 mAbs

reacted with the region between aa 60 and 143; 1 out 3 p30 mAbs reacted with the region between aa 130 and 143. We also

estimate the relative affinity and identified good candidates for diagnostic assay. The fine mapping using commercially

synthesized overlapping oligomers is currently ongoing.

P02

Dynamics of protection against virulent challenge in swine vaccinated with attenuated African Swine

Fever Viruses and their differences in pathogenesis and immune responses

Jolene Carlson, Vivian O’Donnell, Marialexia Alfano, Lauro Velazquez Salinas, Peter Krug, Michael Eschbaumer, Lauren Holinka, Guillermo R. Risatti, Douglas P. Gladue, Stephen Higgs, and Manuel V. Borca

Agricultural Research Service, U.S. Department of Agriculture, Plum Island Animal Disease Center, Greenport, NY 11944, USA. - Biosecurity

Research Institute and Department of Diagnostic Medicine and Pathobiology, College of Veterinary Medicine, Kansas State University,

Manhattan, KS 66506, USA. Department of Pathobiology, University of Connecticut, Storrs, CT 06269, USA.

Keywords: ASFV, Interferon-gamma, Cytokine, Antibody, Protection

African swine fever is a lethal hemorrhagic disease of swine caused by a double-stranded DNA virus. There is no vaccine to

prevent the disease and current control measures are l imited to culling and restricted animal mov ement. Swine infected with

attenuated strains are protected against challenge with homologous virulent virus but there is l imited knowledge of the host

immune mechanisms generating that protection. Swine intramuscularly infected with Pret4 virus develop cl inical disease by

day 3-4 pi ending with all swine dead or euthanized by day 10 pi, while a derivative strain lacking virulence-associated gene

9GL (Pret4 delta9GL virus) is completely attenuated. Groups of swine were infected with Pret4 delta9GL virus and challenged

with the virulent parental virus at 7, 10, 14, 21, and 28 dpi. ASFV-specific IFN-γ production in PBMCs, as well as anti -ASFV

antibodies and cytokines in serum were assessed in each group. Challenge at 7 dpi had 40% surviving while other groups

challenged between 10 and 28 dpi had 60-80% surviving challenge. This model was used to correlate the presence of host

immune response and protection against the challenge. Protection increased with time between first infection and challenge,

with the exception of ASFV-specific antibodies in the surviving swine challenged at 21 and 28 dpi, no solid correlation

between any of the parameters assessed and the extent of protection could be established. These results underscore the

complexity of the system under study where it is very plausible that protection against disease or infection relies heavily on

the concurrence and/or interaction of different host immune mechanisms .

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P03

Comparative study of protection in pigs immunised by different routes and doses with the naturally

attenuated African Swine Fever Virus isolate OUR T88/3 and role of immunomodulatory cytokines.

David Chapman, Pedro J. Sanchez-Cordon, Tamara Jabbar, Ana L. Reis, Lynnette Goatley,

Christopher L. Netherton, Linda Dixon

Keywords: ASFV, Vaccine, Immunity

The present study is an extension of previous works where protection induced by intramuscular administration of the

attenuated OUR T88/3 genotype I isolate was demonstrated. In the work reported here, comparative assessments were

util ised in order to demonstrate differences among various combinations of doses (103, 104 and 105 TCID50/ml) and routes

of immunisation, including the commonly used intramuscular route along with intranasal route. The results showed that

intranasal immunisation of pigs with low and moderate doses (103 and 104 TCID50/ml) provided complete protection (100%)

against lethal challenge with a homologous high virulent ASFV isolate. Moreover protected pigs had either no detectable or

minimal quantities of viral genome in blood at termination. In addition, only mild and transient adverse clinical reactions a nd

mild lesions, consistent with secondary bacterial infections, were observed before a nd after challenge. However, in group of

pigs immunised intranasally with 105 TCID50/ml and in all groups immunised intramuscularly (103, 104 and 105 TCID50/ml);

the rates of protection conferred were lower ranging from 33% to 66%. Regarding immune protection mechanisms, along

with the possible role of humoral immune response, our results suggested that survival of pigs after challenge was associated

with a balance between pro- (TNFα and IL-1β) and anti -inflammatory cytokines (IL-10), without participation of IFNγ. In

contrast, animals that died showing an acute form of ASF displayed an imbalance linked to an exacerbated increase of IL -10

along with an anomalous antibody response.

P04

Evaluation of porcine adenovirus vectored vaccines for African Swine Fever

Shawn Babiuk1, Thang Truong

1, Juanita van Heerden

2, Livio Health

2, Lorne Babiuk

3, Vani Priyadarshini Velpula

4, Amit Gaba

4,

Volker Gerdts4, Andy Potter

4, Suresh K.Tikoo

4

1Canadian Food Inspection Agency, National Centre for Foreign Animal Disease - 2ARC-OVI South Africa - 3University of Alberta - 4Vaccine &

Infectious Disease Organization-International Vaccine Center, University of Saskatchewan, Canada

Keywords: Porcine adenovirus; vaccine, African swine fever

Currently, there is no available vaccine for African swine fever virus (ASFV). Inactivated vaccines as well as subunit vaccines

for ASF have not been able to demonstrate protection against ASF. It has been previously demonstrated by other

laboratories that it is possible to elicit protective immunity against homologous ASF challenge using attenuated live ASF

vaccines. In addition, DNA vaccines which elicit cell mediated immunity have also been demonstrated to provide partial

protection against homologous challenge. These experiments i l lustrate tha t strong cellular immunity is required for a vaccine

to be effective against ASF. Porcine adenovirus vectored vaccines are able to elicit strong cellular immunity and have been

previously demonstrated as an effective vaccine vector in swine. Bioinformatic analysis using different computational

prediction tools were used to identify the potential T and B cell antigens \epitopes of Malawi genotype II ASFV. These

potential antigens or chimeric antigens (containing different epitopes) were expressed using adenov irus vector systems

(porcine adenovirus-3 \ human adenovirus-5) .Three recombinant adenoviruses expressing three vaccine antigens are

currently being evaluated in a homologous ASF Malawi genotype II virus challenge model. Groups of six pigs will be

vaccinated two times with different recombinant adenoviruses. Following vaccination, the pigs will be challenged with

Malawi genotype II and then evaluated for clinical disease and viral loads in blood and swabs from mucosal surfaces.

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P05

Is Ornithodoros erraticus able to transmit the Georgia2007/1 African Swine Fever Virus isolate to

domestic pigs?

Bernard J.1,2

& Vial L. 2 (equal participation), Hutet E.

1, Paboeuf F.

1, Michaud V.

2, Boinas F.

3, Le Potier MF.

1

1Unité Virologie et Immunologie Porcines, Lab. de Ploufragan-Plouzané, ANSES, Univ. Bretagne-Loire, Ploufragan, France. - 2CIRAD, UMR

INRA-CMAEE / 1309 "Contrôle des maladies animales exotiques et émergentes", Campus International de Baillarguet, Montpellier, France. - 3Centre for Interdisciplinary Research in Animal Health (CIISA), Faculty of Veterinary Medicine, Universi ty of Lisbon, Lisbon, Portugal

Keywords: ASF, Tick, vector competence, transmission

As no vaccine or antiviral are available to fight against African swine fever (ASF), the only tools to control it are preventive

measures based on early detection and actual knowledge of the epidemiological risks. In sub -Saharan countries of Africa, ASF

persistence is described to be related to different and complex epidemiological scenarii involving domestic and wild suids and

soft tick vectors of the genus Ornithodoros. In EU, O. erraticus, is known to be able to maintain and/or transmit some ASFV

isolates classified in the genotype I and to amplify the Georgia2007/1 ASFV (genotype II), at least during 3 months. The

objective of the current study was to evaluate the ability of O. erraticus ticks to become infected and to transmit Georgia

2007/1 ASFV to pigs. Ticks were engorged artificially on parafilm membrane and naturally through biting on vireamic pigs. For

artificial feeding, the blood was infected by mixing with an ASFV culture or it was directly sampled on a vireamic pig. The t icks

infected through natural blood feeding were then proposed to secondarily engorge on healthy pigs three and five months

later, respectively, to assess their capacity to transmit the virus. Depending on the infection method, the percentage of

infected ticks and the ASFV titer in ticks were different. The virus load was higher in tick when it was engorged by biting a

vireamic pig than by artificial engorgement except if the blood was sampled on a vireamic pig. The preliminary trial of virus

transmission using ticks previously engorged on vireamic pigs did not allow concluding on the ability of O. erraticus to

transmit Georgia2007/1. Conversely, the inoculation of naturally infected tick homogenate caused typical ASF clinical signs i n

pigs. Further hypotheses will be discussed during the workshop before concluding on transmission.

P06 Research facilitation of pig sector and government preparedness for exotic incursions of swine fevers:

the case of Scotland Thibaud Porphyre

1, Carla Gomes-Correia

2, George J. Gunn

2

1The Roslin Institute, University of Edinburgh, UK - 2Scotland’s Rural College (SRUC), UK

Keywords: Preparedness; Control; Modelling; Industry structure; Exotic pig diseases

The pig industry is one of the major agricultural l ivestock sectors for Great Britain (GB). However, insufficient knowledge

currently exists on the pig sector’s structure and its particular vulnerability to incursions of swine fever (SF). An extensive

programme of research initiated by Scottish Government (SG), through its Centre of Expertise on Animal Disease Outbreak

(EPIC) and its Strategic Research Programme improves the evidence base for rapid disease control decisions in the face of SF

outbreaks. This programme aims to help SG (1) better understand pig industry structure, and (2) develop more robust

surveillance and control plans against SF. Researchers therefore acted as knowledge brokers between industry stakeholders

and policy-makers, allowing for critical issues and limitations to be identified and ground-truthing assumptions and research

results. Overall, the emergent industry structure is more complex than the theoretical top-to-bottom structure of vertically

integrated pig breeding companies. Efforts focussed on risks posed by the non-commercial (i .e. backyard) herds, which

greatly differ in biosecurity and movement behaviours from commercial herds but are connected through animals’

movements, potentially exacerbating spread over wide geographical areas. Integrated modelling study results demonstrate

how widespread epidemics are possible throughout the year, regardless of the duration of the silent period. However, few

geographical areas in GB were likely to generate epidemics from single incursions. The limited influence of behavioural

factors in the uptake of mitigation measures will be presented. This ongoing applied/policy l inked research programme has

already generated invaluable knowledge for developing more robust biosecurity and surveillance plans for emerging/exotic

swine diseases for GB. The predominant benefit, however, has been to facil itate collaborations and discussions between

researchers and various actors for better uptake of coherent industry level exotic swine disease control.

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P07

The role of socio-economic factors on African Swine Fever in Sardinia

Federica Loi, Stefano Cappai, Annamaria Coccollone, Sandro Rolesu

Istituto Zooprofilattico Della Sardegna - Osservatorio Epidemiologico

Keywords: African swine fever, Material Deprivation Index, eradication plan, risk factor

In Sardinia, African swine fever (ASF) has been present since 1978 with a specific epidemiological situation influenced by r isk

factors such as uncontrolled movements of animals and local breeding traditions. Although different control strategies have

been adopted, some socio-cultural risk factors remain present on the island hampering the success of eradication plans. The

recent research activities and the programs in action finalized to ASF control and eradication, have highlighted a strong

association between outbreaks and specific geographic areas, where probably the social and cultural context plays a key role.

Particularly, the il legal pigs breeding is universally recognized like principal risk factor of ASF persistence in region. To

evaluate socio-epidemiological relation between ASF and social condition, number of seropositivity and virus positivity were

evaluate related to Material Deprivation Index (IDM). The deprivation indices are instruments able to indirectly synthesize

the possession of material and social resources connected to geographical different size areas; within these areas the

proportion of people presenting a certain combination of features, indicative of a socio-economic disadvantage condition, is

measured. The IDM has been made for all 377 Sardinian municipalities using last ISTAT 2011census data. The result of the

analyses conducted for each municipality show a strong correlation between the number of ASF seropositivity and virus

positivity and IDM value, indicating an higher concentration of cases in those municipalities of Sardinia that are more

materially deprived. This work will be useful to enrich the a ctual eradication plan with specific socio-cultural actions.

P08

African Swine Fever Risk Assessment and Preparedness in Georgia

I. Menteshashvili , T. Chaligava, O. Parkadze, L. Ninidze, Z. Asanishvili , T. Napetvaridze, N. Kartskhia, D. Khelaia, L. Avaliani

National Food Agency (NFA) of the Ministry of Agriculture (MoA), Tbilisi,

Keywords: ASF, Risk, Assessment, Surveillance

African swine fever (ASF) is a highly contagious viral infection of domestic and wild pigs that is usually lethal and for whi ch no

vaccines exists. Pigs become infected mainly through the oropharyngeal route after contact with infected pigs or through

feeding on contaminated products (e.g. swill and garbage) Transmission through vectors (Ornithodoros ticks) can cause long -

term virus persistence. In April 2007, Georgia reported ASF, with the majority of regions being affected. Various factors

associated with regions at high-risk for ASF include: the density of pig farms, associated level of trade, and/or the presence of

an ASF sylvatic cycle, which could be seasonally dependent. In some areas, domestic animals are in contact with wild hosts

(wild boars, hogs), increasing the risk of transmission. ASF risk assessment was conducted by epidemiologists at NFA, as part

of the national early-warning system emergency diseases. Risk assessment consists of identifying threats, assessing the

likelihood that disease outbreak might occur, and modifying the risks by evaluating their potential consequences. Risk

assessment and analysis is continuous and regularly updated at NFA. Annually, NFA trains 120 government and 200 private

veterinarians at the field level. Training focusses on ASF and the following topics: early detection of the infection; enabli ng

legislation for declaring national emergency measures; surveillance zones and disease free zones; inspection and quarantine

procedures to contain the disease, including pig movement controls and prohibitions on the sale of potentially infected pig

products; epidemiological surveillance and analysis; safe burial or burning of carcasses and other infected material; cleaning

and disinfection of infected premises. NFA devote an appropriate level of resources to implementing effective border and

import quarantine policies that prevent the introduction of ASF diseases in country.

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P09

ASF Public Outreach Program in Georgia

T. Chaligava, L. Avaliani, O. Parkadze, L. Ninidze, Z. Asanishvili , T. Napetvaridze, N. Kartskhia, D. Khelaia, I. Menteshashvili

National Food Agency (NFA) of the Ministry of Agriculture (MoA), Tbilisi, Georgia

Keywords: ASF, outreach campaign, awareness

Due to potential economic implications, African swine fever virus (ASFV) is a pathogen of bioterrorism and veterinary health

concern. Georgia was affected by a nationwide outbreak of ASF in April 2007. ASF outbreaks were simultaneously reported in

three different locations across the country. Reports indicate that the index case had occurred several weeks earlier.

Epidemiological data suggests that the outbreak started in the Poti harbor r egion, probably as a result of direct contact

between free-roaming pigs and insufficiently decontaminated/attenuated waste from ships. The main aim of the public

outreach program was to raise awareness to mitigate future outbreaks of ASF in Georgia. Additi onally, this project

contributed to long-term sustainability by developing a framework for conducting outreach campaigns for future disease

outbreaks. Representatives in Georgia were taught how to conduct and implement an outreach campaign, including how t o

identify target audiences (e.g. local farmers, veterinarians, and members of the pork industry) and effectively create and

disseminate educational materials. Over 450 veterinarians and farmers were trained and 20,000 educational materials (i.e.

flyers, guidelines, etc.) were distributed during the outreach campaign. Pre- and post-ASF knowledge tests were developed

and improvements in trainee knowledge after completing training was observed, post-test scores on average were 16%

higher than pre-test scores; indicating that the outreach program was successful. Regional and state veterinarians, along with

government agencies will be responsible for measuring the long-term success through analysis of ASF lab results, monthly

disease reports, and veterinarian updates. The number of swine disease cases, specifically the number of ASF outbreaks, will

be an indicator of long-term success of the outreach program.

P10 Raising awareness of farmers and veterinarians for efficient respond to African Swine Fever in Ukraine

Datsenko Roman, Nevolko Oleg

Keywords: African swine fever, epidemiology, Ukraine

African swine fever (Pestis Africana suum, ASF, Montgomery’s disease) is a vi ral disease of swine characterized by fever, hem orrhagic

diathesis, significant bleeding, and dystrophic-necrotic changes in various organs. Both domestic and wild pigs are susceptible to ASF. It i s

one of the most dangerous diseases of pigs that becomes widespread in European countries and is mandatory for reporting to th e World

Organization for Animal Health (OIE). Ukra ine reported 65 ASF outbreaks in 2012-2016. Aim. To increase awareness of ASF among

veterinarians and farmers through a public outreach and educational activity. Methods. Workshops to train tra iners on the reg ional level ,

in particular, veterinarians and epidemiologists working at the state animal hospitals and state regional veterinary administrati ons. Results.

In 2015, State Scientific and Research Institute of Laboratory Diagnostics and Veterinary and Sanitary Expertise (SSRILDVSE) and Institute of

Veterinary Medicine (IVM) of the National Academy of Agrarian Sciences took part in the project entitled “Community outreach to support

understanding of African swine fever (ASF) ecology and epidemiology in Eastern Europe (EE): Tra ining and implementation for methods and

strategies for control and prevention” that was implemented within the Defense Threat Reduction Agency (DTRA) Cooperative Bi ologica l

Engagement Program (CBEP) in Ukraine. The project aimed at establishing a regiona l alliance between Armenia, Georgia, Kazakhstan, and

Ukra ine to exchange experience and raise awareness to and provide education on ASF. Specialists of SSRILDVSE and IVM were tra ined as

tra iners before implementing their own outreach program in Ukraine. Th rough this project, 14 tra ining sess ions were arranged in 14

Oblasts of Ukraine. Veterinarians from 307 rayons took part in these tra inings . 531 epizootologis ts of s tate regional veterin ary

administrations and 4482 veterinary doctors of district animal hospitals were tra ined. Additionally, educational materials (flyer and poster)

were developed. They included information about the clinica l and epidemiologica l patterns of ASF, common sources and routes o f

exposure, preventative measures, how to recognize symptoms, and how to respond to suspected ASF cases . 100 000 flyers were printed

and distributed among farmers and populations, as well as 1500 posters for veterinarians of 24 Oblasts. Due to these efforts , necessary

information was brought to the attention of veterinary doctors of district animal hospitals who communicate di rectly with farmers and

persons that work with swine. Conclusion. The ASF outreach and education campaign in Ukraine contributed to effective monitor ing and

reporting of ASF, as well as to ra ising awareness of farmers and population about prevention and control of ASF which in turns makes i t

possible to respond more efficient to ASF outbreaks. Early diagnostics and effective measures can loca l i ze the disease and pr event i ts

further spread i n the country.

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P11

Quality of available habitats for wild boar as a online cartographic tool for managing African Swine

Fever in Eurasia

J. Bosch1; I. Iglesias

1; M.J. Muñoz

1; A. de la Torre

1 ; J. Bosch

1; I. Iglesias

1; M.J. Muñoz

1

1Research Centre in Animal Health, CISA-INIA, Ctra Algete - El Casar s/n, Valdeolmos, 28130 Madrid, Spain.

Keywords: African Swine Fever, surveillance, wild boar distribution, land coverage, habitat

The current African Swine Fever (ASF) epidemic in Eurasia represents a risk for the swine industry with devastating socio-economic and

pol itical consequences. Wild boar appears to be a key factor in maintaining the disease in endemic areas and spreading the di sease across

borders, including within the European Union. Therefore, i t is crucial to know their distribution to help predict and interpret the dynamics

of ASF infection. We present here the online vers ion of the s tandardized dis tribution map based on global land -cover vegetation

(GlobCover) that quantifies the quality of available habitats (QAH) for wild boar across Eurasia through an expert opinion, as an indirect

index for quantifying wild boar. The QAH-map were validated using field data on wild boar locations from several databases as well as the

locations of ASF notifications in wild boar and domestic pigs (WAHID, 2007-2016). This map can serve as a useful epi-tool for defining ri sk

scenarios and identifying potential travel corridors for ASF, which could help manage populations of wild boar and domestic p ig in wildl i fe

management and epidemiological studies. This tool could help in resource a l location decis ions and improve prevention, control and

surveillance of ASF and potentially other diseases affecting swine and wild boar in Eurasia. This map was produced at 300 -m res olution

us ing ArcGis10.0 (ESRI©). The results are available online to the public, providing access to the data and the map for a l l wh o may be

interested on it (researchers, wildlife managers, epidemiologists and adminis tration). This work was supported by FP7/2007-2013 and

AT2015-002 projects .

P12

Integrating ASF sero-prevalence data in wild boar with the decreased-virulence-hypothesis

Laura Meier, Martin Lange, Hans-Hermann Thulke

Helmholtz Centre for Environmental Research - UFZ; Department of Ecological Modelling; PrGr Ecological Epidemiology

Keywords: ASF, serosurvey, seroprevalence, modelling, observational evidence,

Since the entry of ASFV into the Eastern European Union the infection in wild and domestic animals was associated with huge l ethal i ty of

above 90%. ASFV spread in the EU was accompanied with lots of surveillance efforts and monitoring performed in wildlife popul ations of

the affected area. In particular the temporal development of the rate of sero -pos i tive samples from the affected area was under

investigation. Increased sero-prevalence might indicate a possible change in case-fatality due to the infection in wi ld boar compared to

observations short after introduction and in early transmission experiments. Identification of a lterations in lethality of the vi rus infection is

important because existing understanding of perpetuation and persistence of ASFV in wild boar could subsequently become quest ionable.

Moreover, accepted control approaches in domestic pigs may a lso be less adequate once th e dead of infected swine will be less inevitably.

However, there are still debates whether one actually can observe declining case fatality from field survey data likewise accessible from the

affected area in the EU. Therefore, we have implemented a novel spatial transmission model of ASF between the animals of a regional

population of wild-boar. The simulation model reflects transmission events depending on s tochastic spatia l proximity of free ranging

animals. The model was tailored to study different sampling procedures a long with the expans ion of the ASFV affected area; and to

confront these with a lternative scenarios regarding the case fatality of the infection. When comparing the temporal dynamics of the vi rtual

serology surveys it was evident that agre ement with available field observation is most plaus ible without relying on the “decreased

lethality hypothesis”. Finally, we conclude from the model experimentations about suitable sampling protocols to actual ly de monstrate

increase in ASF case fata l i ty us ing serologica l data.

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P13

Evaluation of the efficiency of passive surveillance in the early detection of African Swine Fever in the

wild boar

Vittorio Guberti1, Frank Koenen

2, Si lvia Bellini

3

1Istituto Superiore per la Protezione e la Ricerca Ambientale (ISPRA), ITALY -

2Veterinary and Agrochemical Research Centre (CODA-CERVA),

BELGIUM -3Istituto Zooprofilattico Sperimentale della Lombardia ed Emilia Romagna (IZSLER), ITALY

Keywords: African swine fever, wild boar, surveillance, early detection

African swine fever (ASF) was introduced in the Baltics countries and in Poland in 2014. In such countries, ASF surveillance is performed in

domestic pigs and wild boar and i t is based on active and passive activities. Passive surveillance in wild boar i s carried out by testing for

vi rus and antibodies a ll s ick or found dead individuals whilst active surveillance is performed testing hunted wi ld boar. To evaluate the

efficiency of passive and active surveillance in early detecting ASF, the average ASF infection parameters observed in the Baltic countries

and Poland were used to settle a theoretica l scenario, assuming a 2% (0,02) prevalence.

• ASF Lethal i ty rate: 95% of the infected wi ld boar die in 5 days (0,95/5=0,19 day-1)

• Hunting rate: 40% of the wi ld boar i s hunted in one -year period (0,4/365=0,0011 day-1)

• Dai ly rate at which a wi ld boar found dead is ASF pos i tive: 0,19*0,02=0,0038 day-1

• Dai ly rate at which ASF infected wi ld boar i s hunted: 0,0011*0,02=0,0000219 day-1

The ratio between lethality and hunting rates is: 0,19/0.0000219=8675. It means that the rate at which ASF vi rus kills an inf ected animal i s

8675 times higher in respect to the rate at which a hunter culls a vi rus positive individual. Under the assumption that any dead wild boar i s

immediately retrieved in the forest, the probability of detecting ASF in dead animals is 174 times higher in dead wi ld boar i n respect to

hunted animals (0,0038/0,0000219). Instead, i t appears that only 10% of dead wild boar is found in the forests and in such instance, the

ratio becomes (0,0038*0,1)/0.0000219= 17,4, but even vi rologically testing only 10% of the dead infected wild boar, the daily rate at which

ASF is detected is 17 times higher in comparison with active surveillance. In a population of 1000 wi ld boar, with ASF prevalence of 2%, passive

surveillance based on testing 10% of dead infected animal identifies 1 positive every 2,5 days, whereas testing hunted animal s can detect 1

pos itive every 36 days . In case ASF diagnosis would be performed only by means of serology in hunted wild boar, the da i ly probabi l i ty of

detecting ASF would decrease to 0,00000109day-1 (0,0000219*0,05), s ince only 5% of the infected animals survive to the infection an d

develop antibodies. This means that, vi rologically testing 10% of dead infected animals has 348.6 (0,00038/0,00000109) times more

probabi l i ty of detecting ASF in respect to active survei l lance, based on serology of a l l hunted wi ld boar.

P14

Serological analyses of African Swine Fever in Nigeria, 2006-2015

Wungak Y. S., Ularamu H. G., Lazarus D. D., Luka P. D., Ehizibolo D.O ., ChukwuedoA. A.

National Veterinary Research Institute Vom, Nigeria

Keywords: African swine fever, ELISA, Antibodies, Antigen, Nigeria, Prevalence

African swine fever (ASF) is an important trans -boundary animal disease with a significant socio-economic impact on

subsistence and commercial pig production in Nigeria. Most smallholder pig farmers in Nigeria depen d on this sector as their

main source of l ivelihoods. Improving the control of ASF will ultimately enhance food security and reduce extreme poverty

among smallholder farmers. In this study we present the report of serological analyses of ASF from 2006 -2015. A total of

3168 sera and 92 tissue specimens were collected during outbreaks in Pigs and analysed for ASF virus antibodies and antigen

detection using indirectenzyme-linked immunosorbent assay (I -ELISA), Blocking ELISA and competitive- ELISA (cELISA),

respectively. An overall seroprevalence of 13.89% (95%CI: 12.72 – 15.13) was recorded within this period. The year 2010-

2012 recorded the highest prevalence of 26.70% (95%CI: 23.81 – 29.74), followed by 2012-2013, with 13.10% (95%CI: 10.62 –

15.94), 2006-2007, 8.92% (95%CI: 6.54 – 11.84), 2008- 2009, 8.69% (95%CI: 6.92 – 10.76) and 2014-2015, 4.52% (95%CI: 2.83

– 6.85). ASF antigen was also detected in 31.52% (95%CI: 22.66 – 41.53) of the tissue specimens. This report reveals that ASF

is stil l enzootic in Nigeria. Therefore it is imperative to put in place a comprehensive routine surveillance with an early and

rapid diagnostic system, and also consider the option of compensation in order to achieve control and eradication of ASF in

Nigeria.

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P15

Biosecurity breaches and within-farm virus contamination during an African Swine Fever outbreak on

a medium sized farm in Uganda

Erika Chenais1,2

, Susanna Sternberg-Lewerin2, Sofia Boqvist

2, Lihong Liu

1, Tonny Aliro

3, Charles Masembe

4, Karl Ståhl

1,2.

1National Veterinary Institute, Uppsala, Sweden - 2Swedish University of Agricultural Sciences, Uppsala, Sweden - 3Directorate of Production

and Marketing, Gulu District Local Government, Uganda - 4Makerere University, Kampala, Uganda

Keywords: environmental sampling, farm level biosecurity, low income countries

Uganda is a low-income country with the largest pig population in east Africa. African swine fever (ASF) is endemic with

numerous yearly outbreaks. In the prevailing smallholder subsistence farming systems, farm biosec urity is largely non-

existent. The continuous circulation of ASF in smallholder settings creates biosecurity challenges also for larger farms.

Outbreaks of ASF are largely under-reported and as a consequence the progression of outbreaks are seldom described,

creating lacunae in the epidemiological knowledge of ASF. In this study we investigated and described an outbreak of ASF on

farm level in an endemic area, including investigations of on-farm environmental virus contamination during the outbreak.

The study was carried out on a medium sized pig farm which had 35 adult pigs and 103 piglets or growers at the onset of the

outbreak. All pigs had died or been slaughtered within three months of the outbreak. Informal interviews with farm

representatives were undertaken at the beginning, during the first month, and three months after the outbreak. Biological

samples were taken on days 4 and 34 of the outbreak. ASF was confirmed by presence of ASFV nucleic material from both

these samplings. Environmental samples (soil, manure, water, feed, pig hair) were taken on day 26 of the outbreak. All 35

environmental samples (soil, manure, water, feed, pig hair) were positive for ASFV nucleic material. The ASFV positive

biological samples confirmed earlier epidemiological knowledge and the clinical picture. Breaches and non-compliance in

biosecurity protocol that most l ikely lead to the propagation and within farm spread of the outbreak was revealed, and

confirmed with the results of the environmental samples. These latter results are important for future formulation of advice

in outbreak situations, especially pertaining to outbreaks in endemic countries.

P16

Phylogeography of the African Swine Fever ornithodoros vectors in Southern Africa

Carin Boshoff1,2,3

; Armanda Bastos2; Livio Heath

1

1Agricultural Research Council-Onderstepoort Veterinary Institute, Transboundary Animal Diseases, South Africa - 2University of Pretoria,

Mammal Research Institute, Department of Zoology & Entomology, Pretoria, South Africa - 3Tshwane University of Technology, Department

of Biomedical Sciences, Pretoria, South Africa

Keywords: Ornithodoros, phylogeny, South Africa

African swine fever virus maintenance and transmission dynamics have been intensively studied in Ornithodoros ticks due to

the role that they play in disease epidemiology and in the African sylvatic. However the taxonomic status of ticks of the

Ornithodoros moubata complex is fraught with inconsistencies and the evolutionary history of the group is understudied. We

address this for the southern African region by genetically characterising tick populations from eight game parks within the

ASF control area of South Africa, and from sampling sites in neighbouring Mozambique and Swaziland and comparing these

with previously characterised populations from Zimbabwe, Namibia, Tanzania and Uganda. In common with a previous study,

the 16S rRNA gene phylogeny revealed the presence of three geographically discrete warthog-associated Ornithodoros

l ineages, but uncovered high levels of intra -lineage variation within the southern African region. Phylogenetic analyses

indicated the presence of two topographically distinct haplotypes in the Kruger National Park that have some degree of

overlap in central to northern region. These results suggest historical barriers to gene flow in the largest game park within

the ASF control zone of South Africa, and that tick variation is largely underestimated.

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P17

Socio-economic impact of African Swine Fever outbreaks in smallholder pig systems in four districts

along the Uganda-Kenya border

Noelina Nantima1, Emily Ouma

3, Hikuepi Katjiuongua

2, Michael Ocaido

4, Mike Barongo

4, Michel Dione

3, Anthony Mugisha

4,

Edward Okoth2 and Richard Bishop

2

1Ministry of Agriculture Animal Industry and Fisheries, Entebbe, Uganda - 2International Livestock Research Institute, Nairobi, Kenya - 3International Livestock Research Institute Kampala, Uganda - 4Makerere University, Kampala Uganda

Keywords: African swine fever, economic impact, financial losses, Uganda, Kenya

A study was done to assess the impact of African swine fever (ASF) outbreaks on smallholder pig farmers along the Kenya -

Uganda border. Data was collected using quantitative and qualitative methods. A cross-sectional questionnaire survey was

undertaken among 645 pig keeping household. Two repeated cross -sectional surveys were undertaken after 3 and 6 months

after the main cross-sectional survey involving 120 households. Twenty four focus group discus sions were carried out in 13

vil lages (7 from Kenya and 6 from Uganda) involving 268 pig farmers both men and women. It was found that 8.9% (n=57) of

the households had ASF outbreaks during 2012. There was no significant difference (p>0.001, X2= 2.8) in the proportion of

households affected by ASF in Uganda and Kenya. On average each ASF affected household lost $190.6. The number of pigs

sold during ASF outbreak was 1.33 times more than those that died due to ASF. The prices of pigs during ASF outbreaks

declined by >50%. A very highly significant proportion of households (~83.1%) felt impact due to psychological fear, closure

of markets, failure to restock, pigs sold early and lower sale prices. It was important that appropriate strategies be develo ped

to ameliorate the impacts of ASF to the farmers.

P18 Consultation on a research strategy for the implementation of a regional control program for African

Swine Fever in Africa

Edward Okoth-Abworo1, Mary Louise Penrith

2, Sam Okuthe

3, Hiver Boussini

4

1International Livestock Research Institute and Biosciences East and Central Africa - 2University of Pretoria - 3FAO Emergency Centre for

Trans-boundary Animal Diseases Operations (ECTAD) - 4African Union - InterAfrican Bureau for Animal Resources

Keywords: African swine fever, Research strategy, Disease control

As a follow up to the Consultation workshop on the Regional strategy for the control of African swine fever (ASF) in Africa

held between 10-12 November 2015 in Ouagadougou, Burkina Faso and the validation and adoption of the strategy

document, the BecA-ILRI Hub convened a consultative workshop to develop a research strategy to support the

implementation of an ASF control program in Africa. The aim of the workshop was to identify key knowledge gaps that

require research and that underpin the implementation of the continental ASF control program. The workshop documented

institutional capacity of partners and on-going activities within the institutions on ASF. Participants identified three broad

research pil lars that include: Understanding of ASF epidemiology underpinned by molecular virus characterization, risk and

socio-economics of ASF; Development of diagnostics needed for the disease surveillance and development of vaccines for its

control. A key result of the workshop was the identification of synergistic activities between institutions and the

establishment of new research and development partnerships. The initial consultation is expected to enhance

communication between current joint research activities, initiate search for donor support and inform implementation of

future collaborative research activities aligned to the identified research gaps in order to support local and global ASF con trol

programs.

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P19

Polymerase Cross-Linking Spiral Reaction (PCLSR) for detection of African Swine Fever Virus (ASFV) in

pigs and wild boars

Grzegorz Woźniakowski, Magdalena Frączyk, Andrzej Kowalczyk, Edyta Kozak, Krzysztof Niemczuk, Zygmunt Pejsak

National Veterinary Research Institute, Department of Swine Diseases

Keywords: African swine fever virus, polymerase cross-linking spiral reaction, pigs and wild boars.

African swine fever (ASF) is considered a considerable threat, in the production of pigs worldwide. The ASF aetiological agent – ASFV, i s the

sole member of the Asfivirus genus, belonging to the Asfarvi ridae family. An effective ASF vaccine is not currently aaccessible, thus the only

measures of ASF spread control include, reliable and fast diagnosis. Officia l ly approved, diagnostic methods inc lude, vi rus i solation,

serological assays, including ELISA and immunoperoxidase assay (IPT) and different modifications of the polymerase chain reac tion (PCR).

The spread of the African swine fever vi rus (ASFV) among infected pigs and wild boars, i s curren tly one of the most important facets of

vi rus transmission in eastern Europe. So far, isothermal amplification methods including loop-mediated isothermal ampl i fication (LAMP)

and cross-priming amplification (CPA) have been used as an alternative among ASFV detection methods. However, the main disadvantage

of LAMP and CPA is the high contamination risk and possibility to generate false positive results . Here we present for the fi rs t time the

des ign, development and practical implementation of polymerase cross-linking spiral reaction (PCLSR) for the fast and direct detection of

genetic ASFV material in the blood and sera of infected pigs and wild boars. It has been shown that PCLSR is a rapid, sensiti ve and speci fic

i sothermal method for the detection of ASFV DNA, in directly collected blood or sera from pigs and wild boars. The sensitivity of PCLSR was

s l ightly below the sensitivity of the official, universal probe l ibrary (UPL) real-time PCR. The PCLSR was capable of detecting ASFV DNA in all

examined blood samples, originating from pigs; n=10 and wild boars; n=78. The obtained results were a lso confi rmed by the officia l ly

approved, real-time PCR. The developed new assay might be further used by local and county veterinary officers, hunters or pig farmers ,

for prel iminary ASF diagnosis. The PCLSR as a novel isothermal amplification method might be also designed and used for rapid det ection

of other swine pathogens .

P20

Genomic analysis of Sardinian 26544/OG10 isolate of African Swine Fever Virus

Bacciu Donatella1; Deligios Massimo

2; Sanna Giovanna

1; Madrau Maria Paola

1; Sanna Maria Luisa

1; Dei Giudici Si lvia

1;

Laddomada Alberto1 & Oggiano Annalisa

1

1Istituto Zooprofilattico Sperimentale della Sardegna, 07100 Sassari, Italy -

2University of Sassari, Biomedical Science, 07100 Sassari, Italy

Keywords: African Swine Fever Virus, Next Generation Sequencing, Sardinia, Italy.

African swine fever (ASF) is an extremely contagious viral disease affecting domestic pigs and wild boars, caused by a large

cytoplasmic DNA virus, the African swine fever virus (ASFV), the only member of the Asfarviridae family. ASF is endemic in

sub-Saharan countries and in Sardinia (Italy) and has become more prevalent in Russia and the Caucasus region since its

spread from eastern Africa to Georgia in 2007. Comparative genomic analysis aims to underscore genetic assortment

diversification in distinct viral isolates, to identify deletions and to carry out evolutionary studies. In this work we used Next-

Generation Sequence (NGS) technology to sequence the first complete genome of a Sardinian ASFV p72 genotype I strain,

noted 26544/OG10, isolated in 2010 from domestic pigs. The genome was assembled as a single contig of 182906 bp long, it

contains 165 ORFs and has a 99.80% nucleotide identity to the L60 strain. The complete 26544/OG10 genome sequence was

submitted to GenBank with accession number KM102979. We performed a comparison analysis against the 16 ASFV

genomes available in the database and found that 136 ORFs are present in nine ASFV isolates annotated to date. The most

divergent ORFs codify for uncharacterized proteins such as X69R and DP96R, which have 51.3% and 70.4% nucleotide identity

to the other isolates. A comparison between the Sardinian isolate and the avirulent isolates OURT 88/3, NHV, BA71V was also

carried out. Major variations were found within the multigene families (MGFs) located in the left and right genome regions.

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P21

A universal protocol to generate consensus level genome sequence for African Swine Fever Viruses

S. Baloyi, J van Heerden and L Heath

Transboudary Animal Disease, Onderstepoort Veterinary Institute, Agricultural Research Council, Onderstepoort, South Africa

Keywords: Next generation sequencing (NGS), Zaire

There are twelve complete African swine fever virus (ASFV) genome sequences published. Molecular epidemiology studies on

ASFV isolates have extensively used partial sequence analysis of variable genome regions to determine relationships between

strains. However, whole genome sequencing (WGS) is the route to achieve a proper discrimination required and clarify the

relationship between the ASFV isolates. Previous sequencing protocols had challenges such as those encountered during PCR

amplification and cell culture. The aim of the present study was to test an alternative approach for the detection of ASFV

using a new purification method combined with next-generation sequencing (NGS). In this study nine AFSV isolates of

different genotypes have been chosen and their DNA were sequenced by NGS to obtain WGS. The study sequence data were

compared to published complete genome sequences from GenBank. Whole genome NGS plays an important role in viral

diagnosis, epidemiological investigation and in host-pathogen relationship.

P22 Development of a DNA/LNA optical biosensor for the rapid diagnosis of African Swine Fever

Biagetti M.1, Cuccioloni M.

2, Sebastiani C.

1, Curcio L.

1, Giammariol i M.

1, Bonfi l i L.

2, Angeletti M.

2, Fel i ziani F.

1 De Mia G. M.

1

1Istituto Zooprofilattico Sperimentale dell'Umbria e delle Marche - Perugia (Italy) - 2Scuola di Bioscienze e Medicina Veterinaria – Università

degli Studi di Camerino - Camerino (Italy)

Keywords: African swine fever virus; diagnosis; DNA/LNA optical biosensor;

African swine fever (ASF) is a highly lethal disease affecting the members of the suidae family, including domestic and feral pigs, wild boar,

a frican wild suidae. The disease is highly infectious and is a threat to a ll countries where pig farmi ng i s widespread. Unti l a few years ago

the disease was confined to the countries of sub-Saharan Africa and Sardinia, in recent years the disease has spread to some countries in

the Caucasus and the Russian Federation. Given i ts uncontrollable diffusion, many efforts have been focused on the development of a

rapid diagnostic test to contain any contagion. The purpose of this work is to evaluate the applicability of an optical biose nsor for the rapid

diagnosis of ASF. We immobilized, on the surface of biosensor, two different probes: a ssDNA probe and a chimeric ssDNA/LNA probe. The

inclusion of LNA oligonucleotides in a DNA probe increases the affinity for complementary s trands, as well as both sensitivit y and specificity

for target nucleotides. Furthermore the LNA oligonucleotide can bind the dsDNA in the major groove forming a dsDNA:LNA triplex. The

extraction of DNA from blood of infected animals was perfomed using FTA mini cards . The extracted DNA was directly analyzed o n a

surface biosensor. For verification of the selectivity of the apparatus, 13 di fferent dsDNA samples extracted from blood of experimental ly

infected pigs have been used. The dsDNA extract was denatured to ssDNA in order to a llow the hybridization with the complemen tary

ssDNA probe. In parallel, the same extracts dsDNA were directly tested with ssDNA/LNA probe. After each use, the surface of the biosensor

derivatized with the two types of probes was regenerated by washing with PBS (ssDNA) or with PBS -T (ssDNA/LNA). As a negative control ,

was used an extract dsDNA of a healthy animal and no appreciable signal was detected. Our results proved that the biosensor d erivatized

with the probe ssDNA has poor selectivety for positive samples, 4 out of 13 samples are at the background level, probabl y due to low vi ra l

load and/or the low sensitivity of the probe, whereas the biosensor derivatized with the ssDNA/LNA probe was able to detect a ppreciable

s ignals on the same samples. The limit of detection was of about 150 vi ral genomics copies . Due to th e higher sens i tivi ty of the probe

ssDNA/LNA and the detection capability in real time, the biosensor can provide quick responses on the presence/absence of the vi rus after

s imple extraction procedures. In fact, the ultimate goal of this work i s to perform analysis directly on the farm by connecting the biosensor

to a portable PC without the ampl i fication s tep of the extracted DNA.

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P23

Complete genome sequencing of Sardinian African Swine Fever Virus isolates

Fredrik Granberg1, Claudia Torres i 2, Annal isa Oggiano3, Maja Malmberg1, Carmen Iscaro2, Gian Mario De Mia 2, Sándor Belák1

1Dept of Biomedical Sciences and Veterinary Public Health (BVF), SwedishUniversity of Agricultural Sciences, Uppsala, Sweden - 2Istituto

Zooprofilattico Sperimentale dell’Umbria e delle Marche, Perugia, Italy - 3Istituto Zooprofilattico Sperimentale della Sardegna, Sassari, Italy

Keywords: African swine fever virus; next generation sequencing; full length sequence analysis

Partial sequence analysis of variable genome regions has been extensively used for molecular epidemiological s tudies of the African swine

fever vi rus (AFSV) isolates. By this analysis the Sardinian isolates have been classified as members of the same p72 genotype I with minor

di fferences in the number of tetramer repeats within the B602 gene. Such low level of intra-genotype diversity doesn’t make i t possible to

investigate the genetic relatedness of ASFV outbreaks in Sardinia. To enable comparison at a higher resolution, nine Sardinia n ASFV isolates

col lected from 1978 to 2012 were selected for whole genome sequencing. Six of them were cell culture isolates whereas two were organ

homogenates and one contained erythrocytes enriched from blood by ultracentrifugation. Al l samples , except the erythrocytes , were

treated with RNase and DNase to remove host material prior to DNA extraction and analysis of quality and quantity. The DNA of the nin e

ASFV samples were fragmented, tagged, and sequenced using an Illumina MiSeq. Moreover, the erythrocyte DNA sample had suffici ent

puri ty (ASFV comprised 67.80% of the genetic content as indicated by the MiSEQ data) and quantity (a few micrograms) to allow long-read

sequencing using the Pacific Biosciences (PacBio) RSII platform. Acquired sequencing reads per sample were analyzed and assembled,

performing both a de novo and a mapped assembling. Verification and gap -filling were performed by Sanger sequencing. The nearly enti re

genomes were obtained for a ll the i solates and one of them, the enriched erythrocyte sample col lected in 2008, was completely

sequenced. This genome sequence of 184.842 bp is rather different from the ASFV complete genomes a l ready publ ished in GenBank ,

mostly in the inverted terminal repeats. This genome was annotated by software-aided transfer of annotations . Further analys is are in

progress to obtain whole genome sequences for the remaining samples and to identify biologica l relevant di fferences by compar ative

genomics .

P24

Molecular analysis of African Swine Fever Virus (ASFV) associated with disease outbreaks in Ukraine

during 2012-2015

Nevolko Oleg, Marushchak Lyudmyla, Sushko Mykola.

Keywords: African swine fever, outbreak, PCR, Ukraine

African swine fever (ASF) is one of the most dangerous vi ral diseases of swine. It is characterized by high mortality and causes s igni ficant

economic losses. Initiated in the Caucasus from Georgia and Armenia (2007-2008), ASF has spread rapidly in Eastern Europe (2007-2015),

southern European part of Russian Federation (2008-2011), entered into Ukraine and Belarus (2012-2013), Poland and Baltic States (2014-

2015) and led to the formation of permanent and resistant distribution areas of disadvantage. By May 2016, Ukra ine reported 6 1 ASF

outbreaks in 11 Oblasts. Objectives. The main goal was to s tudy DNA of ASFV isolated from pa thological material selected from domestic

and wild pigs during ASF outbreaks in Zaporizhia, Luhansk, Chernigov, Sumy and Kyiv regions and to compare data on phylogenet ic analysis

of ASF vi rus (ASFV) isolated in Russian Federation, Poland, Baltic and Caucasus. Methods. Samples were collected from a l l locations with

the reported ASF cases: Zaporizhia (2012) – 1 sample, Luhansk (2014) - 5 samples from wild boars and 1 - from a domestic pig; Chernigov

(2014) - 1 sample from a wild boar and 1 sample from a domestic pig, Sumy (2015) - 1 sample from a wild boar, Kyiv (2015) - 1 sample from

the domestic pig. Genomic DNA was amplified by OIE-PCR and qPCR. Genetic characterization of the viral DNA was achieved by sequencing

three independent regions of ASFV genome including: C-terminal end of VP72 coding protein gene, the full genome sequence of the p54-

gene, and the central variable region (CVR) within the B602L-gene. Results of this analysis were compared with phylogenetic data on ASFV

detected in Russia, Poland, Baltic countries, and Caucasus. Results. All samples tested positive by PCR and qPCR assays. PCR protocols with

primers p72-U/p72-D, which produce the amplicon 478 bp, primers PPA89/PPA722 – 678 pb, primers CVR1/CVR2 – 665 pb, recommended

by EU and FAO Reference Laboratory for ASF, (CISA-INIA), Valdeolmos, Madrid, Spain), were used for genotyping. These three independent

regions of the ASFV genome were investigated by sequencing. Phylogenetic tree was generated and analysed. Phylogenetic analys is was

conducted using the Ceneious program. Conclusions. The sequence analysis of VP72, p54, and CVR regions of Ukra inian ASFV genome

exhibited 100% identity to vi ruses registered during ASF outbreaks in Caucasus (2007), Russ ia (2008), Poland (2014), and the Baltic

countries (2014). This genotype of ASFV is responsible for the disease outbreaks in the Eastern Europe s ince i ts introduction in Georgia

(2007) and belongs to the genotype II of ASF vi ruses .

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P25

The challenges encountered in diagnosing African Swine Fever in Uganda: are positives truly positive?

Charles Masembe1, Johnson Mayega

1 and Vincent Muwanika

2

1Makerere University, College of Natural Sciences, School of Biological Sciences, Department of Zoology, Entomology and Fisher ies Sciences,

Uganda - 2Makerere University, College of Agriculture and Env't Sciences, School of Forestry, Geographical and Environmental and Scienc es,

Department of Environmental Management,

Molecular Genetics Laboratory, KAMPALA, Uganda,

Keywords: PCR Diagnosis, False Positive, Realtime, Conventional, Blood Direct, African swine fever

African swine fever (ASF) is a highly contagious infectious disease of pigs, with serious social and economic impacts constra ining trade of

swine and pig products and affecting food security. This disease is a major constraint to piggery in endemic countries. Whenever i t s trikes ,

morta lity rates reach between 90-100% in the affected animals and the disease has neither treatment nor vaccine interventions .

Polymerase Chain Reaction (PCR) is currently considered as a standard test for diagnosis of ASF. Using the PCR method, the sample DNA is

probed for the ASF vi rus genome which confirms presence or absence of the vi rus in the host. Recently some confirmed positive samples

in the laboratory with the Realtime and conventional gel based PCR method have been found negative after sequencing. The full extent of

this problem has not yet been determined. In this study, domestic pig samples were col lected from severa l ASF outbreaks repor ted in

selected districts of Uganda. These samples were tested for ASF using different ASF diagnostic methods both PCR based and one non PCR

based method: the Realtime PCR method, Conventional PCR, Blood Direct PCR method and Immunochromatographic latera l flow test. A

tota l of 77 samples were tested. Preliminary Results indicate that the blood direct PCR method as a more sensitive method com pared to

the Realtime PCR with 48% of the samples testing positive for ASFV. These results highlight a gap in reliability of the d iagnostic methods

currently under use in an endemic setting.

P26

Molecular characterization of Malagasy African Swine Fever Virus strains from 2008 to 2014.

RANDRIAMPARANY Tantely1, MICHAUD Vincent2,4, ALBINA Emmanuel2,3

1LNDV - Laboratoire National de Diagnostic Vétérinaire, Antananarivo, Madagascar - 2CIRAD, UMR CMAEE, Montpellier, France - 3CIRAD,

UMR CMAEE, Petit-Bourg, Guadeloupe, France - 4INRA, UMR1309 CMAEE, Montpellier, France,

Keywords: ASF, PCR, Phylogeny

African swine fever (ASF) is an economically important disease of domestic pigs in the world. ASF is occurred in Europe and

endemic in most of sub-Saharan Africa, including Madagascar Island. Sporadic cases are reported with a 17.8% [95% CI, 10.26

- 25.66] viroprevalence for different ASFV isolates and different areas collected within 2008 -2014 from Madagascar.

Comprehensive relationship between isolates and comparative analyses were performed using public available sequences by

the maximum likelihood method. Isolate genotyping was assessed by comparing concatenated amino acid sequences of

proteins encoded of proteins encoded B646L, KP177R and CP204L genes from 346 (including 15 Malagasy), 140 (including 7

Malagasy), and 37 (including 14 Malagasy) isolates respectively. Analysis of the fragment of the gene B646L showed 100 %

homology among isolates suggesting a single isolate of contamination until now. However, CP204L and KP177R genes

showed more diversity , respectively with 0.9 % and 0.6% difference but no new s patiotemporal affi l iation has been

highlighted that would indicate the origin of new genotype. This stability can be explained notably by the virus circulation

exclusively domestic cycle that follows the virus in the island. Analysis showed that Malagasy i solates studied always placed

in the viral genotype II. However, the diversity among the isolates from Madagascar remains low at these loci despite over 18

years of virus circulation.

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P27

African Swine Fever situation in Lithuania

S. Pileviciene, V. Jurgelevicius, J. Buitkuviene, R. Zagrabskaite, G. Pridotkas

National Food and Veterinary Risk Assessment Institute

Keywords: African swine fever viruse, wild boar, domestic pig, PCR, ELISA, IPT

On 24th January 2014 Lithuania notified two primary cases of African swine fever (ASF) in wild boar: one male animal, 12

months old, hunted on 22 of January 5 km from the border to Belarus and one female, 3 years old found dead on 20 January

2014 about 40 km north from the border to Belarus. The distance between the two animals was about 36 km. The animals

were tested positive for ASF virus (ASFV) genome by real time PCR at the National Reference Laboratory for ASF in Lithuania

(NRL). The results were confirmed by the European Reference Laboratory for ASF (CISA-INIA, Madrid, Spain). Intensive wild

and domestic animal monitoring programme was started. First positive ASF in a large (~20000 pigs were kept) commercial pig

holding with the highest biosecurity confirmed on 23rd of July 2014 in Ignalina district. 290 pigs were sampled from infected

farm, and 102 were found positive. 19 217 pigs were kil led and destroyed by burying on the territory of the farm. And then

ASFV outbreak territory expanded to middle of Lithuania. During this period ~24000 animals were tested for ASF (>10000

domestic pigs, >13000 wild boar). In 2015 were tested 25679 domestic pig (13 were positive) and 24188 wild boar (132 were

positive) and in 2016 til l 1 of June were tested 1887 domestic pig, 13547 wild boar samples (118 were positive). During 201 4-

2015 the Institute has received 32 food samples (meat products) confiscated on the border with Belarus, and found 4 of

these to be positive for ASFV. The methods used for ASFV detection included higly specific real -time PCR, enzyme-linked

immunosorbent assay (ELISA) Ab and Ag, immunoperoxidase test (IPT) (since 2015) and pathological examination.

P28 Validation of new Elisa & PCR for the diagnosis of African Swine Fever

Loic Comtet1, Mickael Roche

1 , Gautier HOURDOIR

2, Séphanie Vérité

1, Fabien Donnet

1, Matthieu LAFFONT

2, Lise GREWIS

2,

Philippe Pourquier1 2

1IDvet, France – 2IDvet Genetics, France

Keywords: ASF, diagnosis, qPCR, serology

African Swine Fever Virus (ASFV) is a highly vi rulent swine disease characterized by fever, hemorrhages and high morta l i ty rates . ASF

control and eradication programs require accurate and reliable diagnostic tests. IDvet offers indirect ELISAs based on three recombinant

ASFV antigens (P32, P62, and P72) for antibody detection in serum, blood filter paper and meat ju ice samples . This ELISA demonstrates

excel lent specificity, especially for wild boar samples. IDvet has a lso developed two new tools, a competitive ELISA and a re a l -time qPCR,

for ASF diagnosis. The ID Screen® African Swine Fever Competition ELISA a llows for the detection of anti-ASFV P32 antibodies . Speci fici ty

was evaluated through the analysis of 280 disease-free sera from domestic and Iberian pigs . Measured speci fici ty was 100.0% (CI 95%:

98.7% - 100.0%). 8 pos itive reference sera from the ASF European Reference Laboratory (EURL-ASF, Madrid, Spain) were correctly

identified as positive. eroconversion was detected between 6 and 13 dpi. The test correctly identi fied sera from a l l genotype s tested,

including genotype II. The test was also evaluated by the EURL. Results indicate a diagnostic specificity of 99.4% (n=177) and a diagnostic

sensitivity of 95.8% (n=213). Perfect agreement (k=0,95) with the IPT (Immunoperoxidase test) was obta ined. The ID GeneTM ASF is a

TaqMan ready-to-use real-time PCR assay based on the simultaneous detection of ASFV and an endogenous internal pos i tive control . It

may be used for blood, serum, plasma, swabs and tissues samples. Results may be obtained in less than two hours (exaction in only 20

minutes, and amplification around 1hour). DNA sample panel from the EURL was tested. The ID GeneTM ASF ki t correctly identi fied a l l

samples (14/14, including DNAs from genotypes I, I I, V, VIII, IX, X) and did not show any cross -reactions with 31 other pathogens . The

detection limit of the PCR was <10 copies, indicating high sensitivi ty. The EURL-ASF reference panel consisting of 16 ASF lyophilised samples

including experimental and clinical field samples, was also tested. DNA extraction was performed by magnetic beads (IDGeneTM Mag Fast)

as per manufacturer’s instructions. Al l samples were correctly scored positive and negative. To conclude, IDvet offers a full ra nge of tools

for the accurate and rapid diagnos is of African Swine Fever, ei ther by serology or qPCR.

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P29

Application of chicken egg yolk anti African Swine Fever viral protein vp73 IgY in a card agglutination

test for sero-diagnosis of African Swine Fever

Mathias Afayoa1, Will iam Olaho-Mukani

3, David Kalenzi Atuhaire

1,2, Boniface Julius Okuni

1, Sylvester Ochwo

1, Kevin

Muwonge1, Kisekka Majid

1, Lonzy Ojok

1

1College of Veterinary Medicine, Animal resources and Bio-security, Makerere University, P.O.BOX 7062 Kampala, Uganda - 2African Union

Centre for Ticks and Tick-Borne Diseases, Private Bag A130, Lilongwe, Malawi - 3African Union Inter-African Bureau of Animal Resources,

P.O. Box 30786, Nairobi, Kenya - 4Corresponding authors: Afayoa Mathias and William Olaho-Mukani

Keywords: African swine fever, IgY, ASF viral proteins, Card agglutination test.

African swine fever (ASF) is highly pathogenic infectious viral haemorrhagic disease of domestic pigs endemic in many African countries

including Uganda, where sporadic outbreaks of the disease occur annually. There i s nei ther vaccine nor drug for ASF; therefor e timely

diagnosis is vi tal for institution of appropriate control measures. The aim of the study was to evaluate the possibility of us ing chi cken egg

yolk anti-ASF vp73 IgY in development of card agglutination test (CAT) for diagnosis of ASF. Purified ASF vi ral protein vp 73 from Ugandan

isolates of ASF vi rus was used to ra ise antibodies in laying hens. Chicken egg yolk immunoglobulin IgY was extracted and puri fied us ing

8.8% NaCl salt precipitation technique and the purified polyclonal antibodies were passively coupled onto aldehyde/Sul fate polystyrene

latex beads suspended in 2-(N-morpholino) ethanesul fonic acid (MES) buffer pH 6.6 and used to detect ASF vi ra l antigens in serum

samples. The diagnostic sensitivity and specificity of positive and negative predictive values o f the developed assay were ca lculated to

eva luate the test performance. The developed in-house CAT had diagnostic sensitivity and speci fici ty of 77.3% and 86.9%, respectively,

whi le the positive and negative predictive values were 74.7% and 88.4% in the same order. In conclus ion, Chicken IgY- based ASF Card

agglutination test could be used in conjunction with other diagnostic tests for field diagnos is of ASF and this could contrib ute towards

control of the disease in East Africa in genera l . However, the developed test should be va l idated further before field appl ication.

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List of participants (List adopted on the 26thof August)

ABDULLINA Valentyna USA [email protected]

METABIOTA, Inc

AFAYOA Mathias Uganda [email protected]

Makerere University

ALONSO Covadonga Spain [email protected]

Instituto Nacional de Investigación y Tecnología Agraria y Alimentaria (INIA)

ATIM Stella Uganda [email protected]

National Animal Disease Diagnosis and Epidemiology Center

AWOSANYA Emmanuel Nigeria [email protected]

Department of Veterinary Public Health and Preventive Medicine, University of Ibadan

BABIUK Shawn Canada [email protected]

Canadian Food Inspection Agency

BASTOS Armanda South Africa [email protected]

University of Pretoria

BELTRAN-ALCRUDO Daniel Hungary [email protected]

Food and Agriculture Organization (FAO) of the United Nations

[email protected] BILOUS Mykola Ukraine

State Service of Ukraine on Food Safety and Customer Protection

Germany [email protected] BLOME Sandra

Friedrich-Loeffler-Institut

USA [email protected] BORCA Manuel

Plum Island Animal Disease Center

BOSHOFF Carin South Africa [email protected]

Agricultural Research Council-Onderstepoort Veterinary Institute

BOURRY Olivier France [email protected]

Anses Ploufragan/ French Agency for food, environmental and occupational health Safety

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CAPPAI Stefano Italia [email protected]

Istituto Zooprofilattico Della Sardegna

CARLSON Jolene USA [email protected]

Plum Island Animal Disease Center

CAVITTE Jean-Charles Belgium [email protected]

European Commission, DG Agriculture and rural development

CHALIGAVA Tengiz Georgia [email protected]

National Food Agency (NFA) of the Ministry of Agriculture (MoA) [email protected]

CHAPMAN David United Kingdom [email protected]

The Pirbright Institute

CHENAIS Erika Sweden [email protected]

National Veterinary Institute, Sweden

COMTET Loic France [email protected]

Idvet

DAS NEVES Carlos Norway [email protected]

Norwegian Veterinary Institute

DATSENKO Roman Ukraine [email protected]

SSRILDVSE

DE LA TORRE Ana Isabel Spain [email protected]

INIA-CISA

DE MIA Gian Mario Italy [email protected]

Istituto Zooprofilattico Sperimentale dell'Umbria e delle Marche (IZS-UM)

DEI GIUDICI Silvia Italy [email protected]

Istituto Zooprofilattico Sperimentale della Sardegna

DELBECQUE Jacques Spain [email protected]

INGENASA

DEPNER Klaus Germany [email protected]


DIXON Linda United Kingdom [email protected]


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DYSHUK Olena Ukraine [email protected]

US Embassy in Ukraine, DTRO-K

ENUKIDZE Tea Georgia [email protected]

Laboratory of the Ministry of Agriculture

ETTER Eric South Africa [email protected]

University Of Pretoria / CIRAD

FILATOV Serhii Ukraine [email protected]

National Scientific Center Institute of Experimental and Clinical Veterinary Medicine

FORTH Jan Hendrik Germany [email protected]

Friedrich-Loeffler-Institute

FRANZONI Giulia Italy [email protected]

University of Sassari / IZS of Sardinia

GALLARDO Carmina Spain [email protected]

Centro de Investigacion en Sanidad Animal

GARRETT Kevin USA [email protected]

Defense Threat Reduction Agency

GAUDREAULT Natasha USA [email protected]

Kansas State University

GAVIER WIDEN Dolorès Sweden [email protected]

National Veterinary Institute (SVA)

GAY Cyril USA [email protected]

USDA Agricultural Research Service

GERILOVYCH Anton Ukraine [email protected]

National Scientific Center Institute of Experimental and Clinical Veterinary Medicine

GLADUE Douglas USA [email protected]


GOGIN Andrei Italy [email protected]

EFSA

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GULBANI Ana Georgia [email protected]


HUTET Evelyne France [email protected]


JAING Crystal USA [email protected]

Lawrence Livermore National Laboratory

JESTIN André France [email protected]


KARGER Axel Germany [email protected]


KEIL Günther Germany [email protected]

FLI/IMVZ

KEßLER Catharina Germany [email protected]

Friedrich Loeffler Institut Federal Research Institute for Animal Health

KOENEN Franck Belgium [email protected]

CODA-CERVA

KOLBASOV Denis Russia [email protected]

National institute for vet virology & microbiology

LANCASTER Mary USA [email protected]


LANGE Martin Germany [email protected]


LE POTIER Marie-Frédérique France [email protected]


LIU Lihong Sweden [email protected]

National Veterinary Institute SVA

LOEFFEN Willie Netherlands [email protected]

Central Veterinary Institute

LOI Federica Italy [email protected]

Istituto Zooprofilattico Della Sardegna

LUKA Pam Nigeria [email protected]

National Veterinary Research Institute,Vom

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LULE Samuel Uganda [email protected]

Mukono District Local Government

MARTINS Carlos Portugal [email protected]

Faculdade de Medicina Veterinaria, Ulisboa

MASEMBE Charles Uganda [email protected]

Makerere University

MAYEGA Johnson Francis Uganda [email protected]

Makerere University

MEIER Laura Germany [email protected]


MENTESHASHVILI Ioseb Georgia [email protected]

National Food Agency (NFA) of the Ministry of Agriculture (MoA)

MICHAUD Vincent France [email protected]

CIRAD

MISINZO Gerald Francis Tanzania [email protected]

Sokoine University of Agriculture

MONTOYA Maria United Kingdom [email protected]


MORROW Alex United Kingdom [email protected]

STAR-IDAZ IRC

MUR Lina USA [email protected]


MURGIA Maria Vittoria USA [email protected]


MUSTRA David Ukraine [email protected]

Metabiota

MWEBE Robert Uganda [email protected]

National Animal Disease Diagnostic and Epidemiology Center

NANTIMA Noelina Uganda [email protected]

Ministry of Agriculture

NEVOLKO Oleg Ukraine [email protected]

SSRILDVSE

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NGELEJA Chanasa Tanzania [email protected]

Tanzania Veterinary Laboratory Agency [email protected]

NIEMCZUK Krzysztof Poland [email protected]

National Veterinary Research Institute, Chief Executive

NURMOJA Imbi Estonia [email protected]

Estonian Veterinary and Food Laboratory

OGGIANNO Annalisa Italy [email protected]

Istituto Zooprofilattico Sperimentale della Sardegna

OGWENG Peter Uganda [email protected]

Makerere University

OKOTH-ABWORO Edward Kenya [email protected]

International Livestock Research Institute

PARKHOUSE Michael Portugal [email protected]

Instituto Gulbenkian de Ciência

PASHYNSKA Vlada Ukraine [email protected]

Science and Technology Center in Ukraine,

PAVLENKO Andrii Ukraine [email protected]

State Service of Ukraine on Food Safety and Customer Protection

PAYNE Ariane Uganda [email protected]

Makerere University

PENRITH Mary Louise South Africa [email protected]

University of Pretoria

PETERSON Melanie USA [email protected]


PIAZZA Valentina Belgium [email protected]

European Commission

PILEVICIENE Simona Lithuania [email protected]

National Food and Veterinary Risk Assessment Institute

PORPHYRE Thibaud United Kingdom [email protected]

The Roslin Institute, University of Edinburgh

REVILLA-NOVELLA Yolanda Spain [email protected]

CBMSO-CSIC

mailto:[email protected]

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RICHT Juergen USA [email protected]


RISATTI Guillermo USA [email protected]

University of Connecticut

RODRIGUEZ Fernando Spain [email protected]

CReSA-IRTA

ROLESU Sandro Italy [email protected]

Istituto Zooprofilattico della Sardegna - osservatorio epidemiologico

ROMAN HERNANDEZ Giselle USA [email protected]


ROSE Nicolas France [email protected]


ROSSI Sophie France [email protected]

Office National de la Chasse et de la Faune Sauvage

ROWLAND Bob USA [email protected]

College of Veterinary Medicine, Kansas State University

RUIZ-FONS Francisco Spain [email protected]

Instituto de Investigación en Recursos Cinegéticos

SALVAT Gilles France [email protected]


SASTRE Patricia Spain [email protected]

INGENASA

SIMON Gaëlle France [email protected]

Anses Ploufragan

SKOROKHOD Serhii Ukraine [email protected]

SSRILDVSE

SKORUPSKI Wlodzimierz Poland [email protected]

Glowny Inspektorat Weterynarii

SMIETANKA Krzysztof Poland [email protected]

National Veterinary Research Institute

SPARGO Reverend Zimbabwe [email protected]

Department of Livestock and Veterinary Services

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STAHL Karl Sweden [email protected]

National Veterinary Institute, SVA

STEINAA Lucilla Kenya [email protected]

International Livestock Research Institute

SYTIUK Mykola Ukraine [email protected]

Institute of Veterinary Medicine

THULKE Hans-Hermann Germany [email protected]


TIGNON Marylène Belguim [email protected]

CODA CERVA

TSOKANA Constantina Greece [email protected]

Research Committee, University of Thessaly

TUMUSIINE Dan Uganda [email protected]

National Animal Disease Diagnostics and Epidemiology Centre,

ULARAMU Hussaini Gulak Nigeria [email protected]

National Veterinary Research Institute Vom

URNIZA HOSTENCH Alicia Spain [email protected]

Zoetis Manufacturing & Research Spain, S.L.

VAN HEERDEN Juanita South Africa [email protected]

Agricultural Research Councii-Onderstepoort Veterinary lnstitute

VEPKHVADZE Nino Georgia [email protected]


VIAL Laurence France [email protected]

CIRAD

VIAPLANA FLORENSA Elisenda Spain [email protected]

Zoetis Manufacturing & Research Spain, S.L.

VILTROP Arvo Estonia [email protected]

Estonian University of Life Sciences

WALZER Chris Austria [email protected]

FIWI - University of Veterinary Medicine Vienna

WENDLING Sébastien France [email protected]

DGAL

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WILLIAMS David Australia [email protected]

CSIRO, Australian Animal Health Laboratory

WOOLAWAY Kathryn United Kingdom [email protected]

DEFRA

WUNGAK Yiltawe Simwal Nigeria [email protected]

National Veterinary Research Institute

YONA Clara Tanzania [email protected]

Sokoine University of Agriculture

ZANI Laura Germany [email protected]


ZARDIASHVILI Tamila Georgia [email protected]

CH2M HILL

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